Virucidal Efficacy of Glutaraldehyde against Enteroviruses Is Related to the Location of Lysine Residues in Exposed Structures of the VP1 Capsid Protein

Glutaraldehyde (GTA) is a potent virucidal disinfectant whose exact mode of action against enteroviruses is not understood. Earlier reports showed that GTA reacts preferentially with the VP1 capsid protein of echovirus 25 and poliovirus 1 and that GTA has affinity for exposed lysine residues on proteins. To investigate further the inactivation of enteroviruses by GTA, seven strains were selected on the basis of differences in their overall number and the positions of lysine residues in the amino acid sequences of the VP1 polypeptide. Inactivation kinetics experiments were performed with 0.10% GTA. The viruses grouped into three clusters and exhibited significantly different levels of sensitivity to GTA. The results were analyzed in the light of current knowledge of the three-dimensional structure of enteroviruses and the viral life cycle. The differences observed in sensitivity to GTA were related to the number of lysine residues and their locations in the VP1 protein. The overall findings suggest that the BC and DE loops, which cluster at the fivefold axis of symmetry and are the most exposed on the outer surface of the virions, are primary reactive sites for GTA.     

Activity of glutaraldehyde at low concentrations against capsid proteins of poliovirus type 1 and echovirus type 25.

The activity of glutaraldehyde (GTA) against capsid proteins of poliovirus type 1 and echovirus type 25 was studied to understand the mode of action of this reagent against enteroviruses. The viruses were treated with GTA concentrations ranging from 0.005 to 0.10%. In the poliovirus particles, high-molecular-weight products were formed by 0.05% GTA, whereas in the echovirus particles, they were formed at 0.005% GTA. These products consist of complexes composed essentially of VP1 and VP3. There seemed to be differences in the composition of the complexes in the two viruses. Cross-linkings between the two polypeptides of the poliovirus capsid may be due to the accessibility to GTA of lysine residues on the loops of VP1 and VP3, which twist out from the surface of the shell.     

Activity of glutaraldehyde at low concentrations against capsid proteins of poliovirus type 1 and echovirus type 25.

The activity of glutaraldehyde (GTA) against capsid proteins of poliovirus type 1 and echovirus type 25 was studied to understand the mode of action of this reagent against enteroviruses. The viruses were treated with GTA concentrations ranging from 0.005 to 0.10%. In the poliovirus particles, high-molecular-weight products were formed by 0.05% GTA, whereas in the echovirus particles, they were formed at 0.005% GTA. These products consist of complexes composed essentially of VP1 and VP3. There seemed to be differences in the composition of the complexes in the two viruses. Cross-linkings between the two polypeptides of the poliovirus capsid may be due to the accessibility to GTA of lysine residues on the loops of VP1 and VP3, which twist out from the surface of the shell.     

Comparative sensitivities of Sabin and Mahoney poliovirus type 1 prototype strains and two recent isolates to low concentrations of glutaraldehyde.

Significant intratypic differences in the glutaraldehyde (GTA) sensitivity of echovirus isolates have been shown. While exploring ways to optimize the study of GTA sensitivity of enteroviruses, we also observed intratypic differences in poliovirus type 1 isolates collected in France. A suspension procedure was used for assessing the virucidal effect of GTA at low concentrations (< or = 0.10%) against purified viruses. Two recent isolates of poliovirus type 1 tested were first fully characterized by the PCR restriction fragment length polymorphism (RFLP) test. The RFLP pattern of clinical isolate 5617 was similar to that of poliovirus type 1 LS-c, 2ab (Sabin strain), confirming the vaccine origin of strain 5617. The RFLP pattern of strain 5915 recovered from sewage was different from that of the Mahoney strain, suggesting a genetic variation in this wild isolate. We then analyzed under the same controlled conditions the GTA sensitivities of both isolates and their respective prototype strains. The wild Mahoney and 5915 strains exhibited significantly lower sensitivities to GTA than did the vaccine Sabin and 5617 strains. The inactivation rates of clinical isolates 5617 and 5915 were very similar to those of their corresponding reference Sabin and Mahoney strains. Both the conformational structure of the capsid of each strain and the amino acid constitution of structural polypeptides could be involved in the variations observed. The relevance of our comparative sensitivity studies to standardization of virucidal tests is discussed.     

Enterovirus 71 Binding to PSGL-1 on Leukocytes: VP1-145 Acts as a Molecular Switch to Control Receptor Interaction

Some strains of enterovirus 71 (EV71), but not others, infect leukocytes by binding to a specific receptor molecule: the P-selectin glycoprotein ligand-1 (PSGL-1). We find that a single amino acid residue within the capsid protein VP1 determines whether EV71 binds to PSGL-1. Examination of capsid sequences of representative EV71 strains revealed that the PSGL-1-binding viruses had either a G or a Q at residue 145 within the capsid protein VP1 (VP1-145G or Q), whereas PSGL-1-nonbinding viruses had VP1-145E. Using site-directed mutagenesis we found that PSGL-1-binding strains lost their capacity to bind when VP1-145G/Q was replaced by E; conversely, nonbinding strains gained the capacity to bind PSGL-1 when VP1-145E was replaced with either G or Q. Viruses with G/Q at VP1-145 productively infected a leukocyte cell line, Jurkat T-cells, whereas viruses with E at this position did not. We previously reported that EV71 binds to the N-terminal region of PSGL-1, and that binding depends on sulfated tyrosine residues within this region. We speculated that binding depends on interaction between negatively charged sulfate groups and positively charged basic residues in the virus capsid. VP1-145 on the virus surface is in close proximity to conserved lysine residues at VP1-242 and VP1-244. Comparison of recently published crystal structures of EV71 isolates with either Q or E at VP1-145 revealed that VP1-145 controls the orientation of the lysine side-chain of VP1-244: with VP1-145Q the lysine side chain faces outward, but with VP1-145E, the lysine side chain is turned toward the virus surface. Mutation of VP1-244 abolished virus binding to PSGL-1, and mutation of VP1-242 greatly reduced binding. We propose that conserved lysine residues on the virus surface are responsible for interaction with sulfated tyrosine residues at the PSGL-1 N-terminus, and that VP1-145 acts as a switch, controlling PSGL-1 binding by modulating the exposure of VP1-244K.     

Sequence and structure of human rhinoviruses reveal the basis of receptor discrimination

The sequences of the capsid protein VP1 of all minor receptor group human rhinoviruses were determined. A phylogenetic analysis revealed that minor group HRVs were not more related to each other than to the nine major group HRVs whose sequences are known. Examination of the surface exposed amino acid residues of HRV1A and HRV2, whose X-ray structures are available, and that of three-dimensional models computed for the remaining eight minor group HRVs indicated a pattern of positively charged residues within the region, which, in HRV2, was shown to be the binding site of the very-low-density lipoprotein (VLDL) receptor. A lysine in the HI loop of VP1 (K224 in HRV2) is strictly conserved within the minor group. It lies in the middle of the footprint of a single repeat of the VLDL receptor on HRV2. Major group virus serotypes exhibit mostly negative charges at the corresponding positions and do not bind the negatively charged VLDL receptor, presumably because of charge repulsion.     

Effect of hypertonic conditions on protein synthesis in cells productively infected with simian virus 40.

Hypertonic medium selectively suppressed the synthesis of most host cell polypeptides relative to the synthesis of simian virus 40 capsid polypeptides and a minority of cellular polypeptides, notably histones. Under optimal hypertonic conditions, the synthesis of the major capsid polypeptide (VP1) is enhanced about sevenfold relative to host polypeptide synthesis. Because of the small amounts of the other nonhistone capsid polypeptides (VP2) and VP3) present in cell lysates, it was difficult to quantitate the extent, if any, of their enhancement. The maintenance of the restricted pattern of protein synthesis caused by hypertonic medium was dependent on continual peptide chain initiations. The resistance of viral protein synthesis to hypertonic conditions provides a means of detecting relatively low levels of intracellular viral protein synthesis. Analysis of the specific activity of the acid-soluble [3H]lysine pool indicated that the rate of incorporation of [3H]lysine into protein was an overestimation of the actual rate of overall protein synthesis occurring in cells exposed to hypertonic as compared to isotonic conditions. Since it is likely that both cellular and viral protein synthesis draw lysine from a single pool, this change in pool specific activity does not affect the analysis of relative rates of protein synthesis at a given level of tonicity.     

Active residues and viral substrate cleavage sites of the protease of the birnavirus infectious pancreatic necrosis virus

The polyprotein of infectious pancreatic necrosis virus (IPNV), a birnavirus, is processed by the viral protease VP4 (also named NS) to generate three polypeptides: pVP2, VP4, and VP3. Site-directed mutagenesis at 42 positions of the IPNV VP4 protein was performed to determine the active site and the important residues for the protease activity. Two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pVP2-VP4 and the VP4-VP3 junctions. Wild-type activity at the pVP2-VP4 junction and a partial block (with an alteration of the cleavage specificity) at the VP4-VP3 junction were observed when replacement occurred at histidines 547 and 679. A similar observation was made when aspartic acid 693 was replaced by leucine, but wild-type activity and specificity were found when substituted by glutamine or asparagine. Sequence comparison between IPNV and two birnavirus (infectious bursal disease virus and Drosophila X virus) VP4s revealed that serine 633 and lysine 674 are conserved in these viruses, in contrast to histidines 547 and 679. The importance of serine 633 and lysine 674 is reminiscent of the protease active site of bacterial leader peptidases and their mitochondrial homologs and of the bacterial LexA-like proteases. Self-cleavage sites of IPNV VP4 were determined at the pVP2-VP4 and VP4-VP3 junctions by N-terminal sequencing and mutagenesis. Two alternative cleavage sites were also identified in the carboxyl domain of pVP2 by cumulative mutagenesis. The results suggest that VP4 cleaves the (Ser/Thr)-X-Ala / (Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.     

Studies on the Classification of Bovine Enteroviruses

Bovine enteroviruses isolated from cattle and other ruminants in various areas of the world were classified into three distinct serotypes by cross-neutralization tests using criteria established for the differentiation of human enteroviruses. According to Western blot analysis, however, immunodominant structural polypeptides VP1 of the viruses tested have common epitopes, recognized by antisera to each of the three serotypes. These findings indicate that non-neutralizing epitopes on VP1 are generally conserved. It is, therefore, conjectured that bovine enteroviruses were derived from a common ancestor.     

Crystal structure of the kringle 2 domain of tissue plasminogen activator at 2.4-A resolution.

The crystal structure of the kringle 2 domain of tissue plasminogen activator was determined and refined at a resolution of 2.43 A. The overall fold of the molecule is similar to that of prothrombin kringle 1 and plasminogen kringle 4; however, there are differences in the lysine binding pocket, and two looping regions, which include insertions in kringle 2, take on very different conformations. Based on a comparison of the overall structural homology between kringle 2 and kringle 4, a new sequence alignment for kringle domains is proposed that results in a division of kringle domains into two groups, consistent with their proposed evolutionary relation. The crystal structure shows a strong interaction between a lysine residue of one molecule and the lysine/fibrin binding pocket of a noncrystallographically related neighbor. This interaction represents a good model of a bound protein ligand and is the first such ligand that has been observed in a kringle binding pocket. The structure shows an intricate network of interactions both among the binding pocket residues and between binding pocket residues and the lysine ligand. A lysine side chain is identified as the positively charged group positioned to interact with the carboxylate of lysine and lysine analogue ligands. In addition, a chloride ion is located in the kringle-kringle interface and contributes to the observed interaction between kringle molecules.     

Characterization of the major iron-regulated protein of Neisseria gonorrhoeae and Neissereria meningitidis

The major iron-regulated protein (MIRP) was purified, from both Neisseria gonorrhoeae and N. meningitidis by selective extraction with cetyltrimethylammonium bromide followed by ion-exchange and moleculair-seive chromatography. Solutions of the purified proteins had a characteristic pink color. The overall amino acid composition of these proteins was similar, although differences were noted in the number of serine, threonine, and lysine residues. Nevertheless, the N-terminal amino acid sequence was identical through 47 residues for both the meningococcal and gonococcal MIRP. Plasma emission spectrophotometry revealed that the meningococcal 37K protein contained ca. 1 mole Fe/mole protein.     

Type-specific and cross-reactive antigenicity of capsid proteins VP1 and VP2 of echovirus type 7

After disruption of echovirus type 7 virions with urea and heat, VP1 and VP2 were separated by isoelectric focusing in urea-containing sucrose gradients. Antisera to these two polypeptides were produced in guinea pigs. In complement fixation, antiserum to VP1 reacted with native and heated virions (N and H antigens, respectively) of homologous virus, and also cross-reacted with heated virions of some other enteroviruses used. Antiserum to VP2 was reactive only with heated virions of homologous and heterologous viruses. Interestingly, the anti-VP2 serum reacted neither with native nor even with heated procapsids (naturally-occurring empty capsids). Antiserum to VP1, but not VP2, showed neutralizing and hemagglutination-inhibiting activities. These results suggest that 1) both VP1 and VP2 possess cross-reactive antigenic determinants which are exposed on the surface of heated virions, and 2) type-specific determinants of VP1 are located on the surface of native virions.     

Symmetry-related clustering of positive charges is a common mechanism for heparan sulfate binding in enteroviruses

Coxsackievirus A9 (CAV9), a member of the Picornaviridae family, uses an RGD motif in the VP1 capsid protein to bind to integrin αvβ6 during cell entry. Here we report that two CAV9 isolates can bind to the heparan sulfate/heparin class of proteoglycans (HSPG). Sequence analysis identified an arginine (R) at position 132 in VP1 in these two isolates, rather than a threonine (T) as seen in the nonbinding strains tested. We introduced a T132R substitution into the HSPG-nonbinding strain Griggs and recovered infectious virus capable of binding to immobilized heparin, unlike the parental Griggs strain. The known CAV9 structure was used to identify the location of VP1 position 132, 5 copies of which were found to cluster around the 5-fold axis of symmetry, presumably producing a region of positive charge which can interact with the negatively charged HSPG. Analysis of several enteroviruses of the same species as CAV9, Human enterovirus B (HEV-B), identified examples from 5 types in which blocking of infection by heparin was coincident with an arginine (or another basic amino acid, lysine) at a position corresponding to 132 in VP1 in CAV9. Together, these data show that membrane-associated HSPG can serve as a (co)receptor for some CAV9 and other HEV-B strains and identify symmetry-related clustering of positive charges as one mechanism by which HSPG binding can be achieved. This is a potentially powerful mechanism by which a single amino acid change could generate novel receptor binding capabilities, underscoring the plasticity of host-cell interactions in enteroviruses.     

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine.     

Reductive alkylation of ribosomes as a probe to the topography of ribosomal proteins

Escherichia coli ribosomes were treated with a number of different aldehydes of various sizes in the presence of NaBH(4). After incorporation of either (3)H or (14)C, the ribosomal proteins were separated by two-dimensional polyacrylamide-gel electrophoresis and the extent of alkylation of the lysine residues in each protein was measured. The same pattern of alkylation was observed with the four reagents used, namely formaldehyde, acetone, benzaldehyde and 3,4,5-trimethoxybenzaldehyde. Every protein in 30S and 50S subunits was modified, although there was considerable variation in the degree of alkylation of individual proteins. A topographical classification of ribosomal proteins is presented, based on the degree of exposure of lysine residues. The data indicate that every protein of the ribosome has at least one lysine residue exposed at or near the surface of the ribonucleo-protein complex.     

Study of heat denaturation of human serum albumin in water-alcohol and water-salt solutions in the presence of organic ligands

The method of differential scanning microcalorimetry was used to show a decrease in heat stability of serum albumin in the presence of aliphatic alcohols. In aqueous-alcohol media, the melting temperature, denaturation transition enthalpy were decreased, and the protein intermolecular aggregation enhanced. When the alcohol concentration in aqueous solution was elevated, the number of epsilon-amino groups of lysine residues in human serum albumin exposed to the solvent rose from 6-7 in aqueous solution to maximum 20 groups in the aqueous-alcohol solution, respectively. The elevation of ionic strength also induced an increase in the number of exposed lysine residues and was accompanied by an enhancement of protein aggregation. The modification of six amino groups by pyridoxal phosphate or three by glucose in the initial albumin stabilized the protein incubated at 65 degrees-70 degrees C both in the aqueous-alcohol media. At the given concentration and temperature the native protein was denatured and fully aggregated. Aliphatic alcohols displaced fatty acids from the binding sites on the molecule of serum albumin, which resulted in a change in the number of peaks of the melting curve.     

A beta-stranded motif drives capsid protein oligomers of the parvovirus minute virus of mice into the nucleus for viral assembly

The determinants of nuclear import in the VP-1 and VP-2 capsid proteins of the parvovirus minute virus of mice strain i (MVMi) synthesized in human fibroblasts were sought by genetic analysis in an infectious plasmid. Immunofluorescence of transfected cells revealed that the two proteins were involved in cooperative cytoplasmic interactions for nuclear cotransport. However, while VP-1 translocated regardless of extension of deletions and did not form capsid epitopes by itself, VP-2 seemed to require cytoplasmic folding and the overall conformation for nuclear transport. The sequence (528)KGKLTMRAKLR(538) was found necessary for nuclear uptake of VP-2, even though it was not sufficient to confer a nuclear localization capacity on a heterologous protein. In the icosahaedral MVMi capsid, this sequence forms the carboxy end of the amphipathic beta-strand I (betaI), and all its basic residues are contiguously positioned at the face that in the unassembled subunit would be exposed to solvent. Mutations in singly expressed VP-2 that either decrease the net basic charge of the exposed face (K530N-R534T), perturb the hydrophobicity of the opposite face (L531E), or distort the betaI conformation (G529P) produced cytoplasmic subviral oligomers. Particle formation by betaI mutants indicated that the basic residues clustered at one face of betaI drive VP oligomers into the nucleus preceding and uncoupled to assembly and that the nuclear environment is required for MVMi capsid formation in the infected cell. The degree of VP-1/VP-2 transport cooperativity suggests that VP trimers are the morphogenetic intermediates translocating through the nuclear pore. The results support a model in which nuclear transport signaling preserves the VP-1/VP-2 stoichiometry necessary for efficient intranuclear assembly and in which the beta-stranded VP-2 nuclear localization motif contributes to the quality control of viral morphogenesis.     

Role of lysine methylation in the activities of elongation factor 1 alpha

Previous work in our laboratory has demonstrated that 19% of the lysine residues in the protein synthesis elongation factor (EF-1 alpha) are methylated when the factor is purified from the mycelial form of the fungus Mucor racemosus. However, the same factor, when purified from spores of M. racemosus, is largely unmethylated. Despite its wide-spread occurrence in a great number of basic proteins, the functional significance of lysine N-methylation remains poorly understood. Spore and mycelial forms of EF-1 alpha were therefore compared in a series of assays to determine their relative affinities for various substrates and cofactors known to interact with the factor during the elongation cycle. The results suggested that hypomethylated and fully methylated EF-1 alpha had equal affinities for GTP, aminoacyl-tRNA, and ribosomes. Also, methylation did not appear to affect the accuracy of translation in an in vitro system. However, experiments did suggest that methylation may affect the ability of the factor to form complexes with other subunits (EF-1 beta gamma) which are known to enhance the overall rate of protein synthesis.     

Sites of protein-protein interaction on the mitochondrial F1-ATPase inhibitor protein.

We have investigated the structure of the mitochondrial F1-ATPase inhibitor protein from ox heart by using a differential trace-labelling method. This method has also been used to determine sites on the inhibitor protein involved in binding to both the isolated mitochondrial ATPase (F1) and to a specific anti-inhibitor antibody. Native, free inhibitor was trace-labelled on its lysine and serine residues with [14C]acetic anhydride, and inhibitor protein unfolded in guanidinium chloride or specifically bound to another protein, with [3H]acetic anhydride. Exposure/concealment of residues was deduced from the 14C/3H ratios of the peptides in a proteolytic digest of the inhibitor, after separation by h.p.l.c. None of the lysine or serine residues in the native inhibitor are as exposed as in the unfolded form. There is a gradient of reactivity, with residues 54-58 being most concealed and exposure increasing towards either end of the protein. A slight decrease in reactivity is noted in residues 1-3, suggesting that the N-terminus may be in a fairly restricted environment. These findings are discussed in the light of the predicted structure of the inhibitor protein. All but one of the labelled residues increases in reactivity when inhibitor protein binds to F1. The exception, Lys-24, is only slightly concealed. Hence, F1 binding appears not to involve the lysine or serine residues directly. This finding is consistent with the view that the F1-inhibitor interaction is hydrophobic in nature. Complementary information was provided using an anti-inhibitor antibody that binds to a site on the inhibitor different from that at which F1 binds. Binding of this antibody conceals residues 54, 58, and 65 considerably. This confirms that F1 does not interact with these hydrophilic residues on the inhibitor protein.     

Crystal structure of the cyclophilin-like domain from the parasitic nematode Brugia malayi.

Cyclophilins are a family of proteins that exhibit peptidyl-prolyl cis-trans isomerase activity and bind the immunosuppressive agent cyclosporin A (CsA). Brugia malayi is a filarial nematode parasite of humans, for which a cyclophilin-like domain was identified at the N-terminal of a protein containing 843 amino acid residues. There are two differences in sequence in the highly conserved CsA binding site: A histidine and a lysine replace a tryptophan and an alanine, respectively. The crystal structure of this domain has been determined by the molecular replacement method and refined to an R-factor of 16.9% at 2.15 A resolution. The overall structure is similar to other cyclophilins; however, major differences occur in two loops. Comparison of the CsA binding site of this domain with members of the cyclophilin family shows significant structural differences, which can account for the reduced sensitivity of the Brugia malayi protein to inhibition by CsA.