Strength of envelope protein interaction modulates cytopathicity of measles virus

To understand the molecular determinants of measles virus (MV) cytopathicity, we have characterized mutant viruses exhibiting a more-extensive cell-to-cell fusion while maintaining efficient replication to high titers. A virus which is modified by the addition of an 8-amino-acid Flag epitope tag at the cytoplasmic tail of its H (for MV hemagglutinin) envelope glycoprotein replicates efficiently, has an increased cytopathicity, possesses a greater infectivity per particle, and has an altered protein composition compared with that of unmodified MV. The mutant phenotype is not specifically linked to the epitope sequence, since an alternatively added HA (for influenza virus-derived hemagglutinin) epitope tag caused similar effects. We demonstrate that both epitope tags weaken the interaction between the H and fusion (F) glycoproteins in virus-infected cells. This reduction in strength of H/F interaction is independent of the presence of the viral matrix (M) protein. Viruses with this less stable complex are more sensitive to neutralization by a soluble octameric form of the CD46 receptor, consistent with their increased fusogenicity. Similar analyses of glycoproteins derived from MV strains with reduced cytopathicities confirm that the strength of H and F glycoprotein interaction is a modulator of viral fusogenicity.     

Analysis of a neutralizing antibody for human herpesvirus 6B reveals a role for glycoprotein Q1 in viral entry

Human herpesvirus 6 (HHV-6) is a T cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6A and HHV-6B, based on differences in their genetic, antigenic, and growth characteristics and cell tropisms. The function of HHV-6B should be analyzed more in its life cycle, as more than 90% of people have the antibodies for HHV-6B but not HHV-6A. It has been shown that the cellular receptor for HHV-6A is human CD46 and that the viral ligand for CD46 is the envelope glycoprotein complex gH/gL/gQ1/gQ2; however, the receptor-ligand pair used by HHV-6B is still unknown. In this study, to identify the glycoprotein(s) important for HHV-6B entry, we generated monoclonal antibodies (MAbs) that inhibit infection by HHV-6B. Most of these MAbs were found to recognize gQ1, indicating that HHV-6B gQ1 is critical for virus entry. Interestingly, the recognition of gQ1 by the neutralizing MAb was enhanced by coexpression with gQ2. Moreover, gQ1 deletion or point mutants that are not recognized by the MAb could nonetheless associate with gQ2, indicating that although the MAb recognized the conformational epitope of gQ1 exposed by the gQ2 interaction, this epitope was not related to the gQ2 binding domain. Our study shows that HHV-6B gQ1 is likely a ligand for the HHV-6B receptor, and the recognition site for this MAb will be a promising target for antiviral agents.     

Minute virus of mice, a parvovirus, in complex with the Fab fragment of a neutralizing monoclonal antibody

The structure of virus-like particles of the lymphotropic, immunosuppressive strain of minute virus of mice (MVMi) in complex with the neutralizing Fab fragment of the mouse monoclonal antibody (MAb) B7 was determined by cryo-electron microscopy to 7-A resolution. The Fab molecule recognizes a conformational epitope at the vertex of a three-fold protrusion on the viral surface, thereby simultaneously engaging three symmetry-related viral proteins in binding. The location of the epitope close to the three-fold axis is consistent with the previous analysis of MVMi mutants able to escape from the B7 antibody. The binding site close to the symmetry axes sterically forbids the binding of more than one Fab molecule per spike. MAb as well as the Fab molecules inhibits the binding of the minute virus of mice (MVM) to permissive cells but can also neutralize MVM postattachment. This finding suggests that the interaction of B7 with three symmetry-related viral subunits at each spike hinders structural transitions in the viral capsid essential during viral entry.     

Functional and Structural Characterization of Neutralizing Epitopes of Measles Virus Hemagglutinin Protein

Effective vaccination programs have dramatically reduced the number of measles-related deaths globally. Although all the available data suggest that measles eradication is biologically feasible, a structural and biochemical basis for the single serotype nature of measles virus (MV) remains to be provided. The hemagglutinin (H) protein, which binds to two discrete proteinaceous receptors, is the major neutralizing target. Monoclonal antibodies (MAbs) recognizing distinct epitopes on the H protein were characterized using recombinant MVs encoding the H gene from different MV genotypes. The effects of various mutations on neutralization by MAbs and virus fitness were also analyzed, identifying the location of five epitopes on the H protein structure. Our data in the present study demonstrated that the H protein of MV possesses at least two conserved effective neutralizing epitopes. One, which is a previously recognized epitope, is located near the receptor-binding site (RBS), and thus MAbs that recognize this epitope blocked the receptor binding of the H protein, whereas the other epitope is located at the position distant from the RBS. Thus, a MAb that recognizes this epitope did not inhibit the receptor binding of the H protein, rather interfered with the hemagglutinin-fusion (H-F) interaction. This epitope was suggested to play a key role for formation of a higher order of an H-F protein oligomeric structure. Our data also identified one nonconserved effective neutralizing epitope. The epitope has been masked by an N-linked sugar modification in some genotype MV strains. These data would contribute to our understanding of the antigenicity of MV and support the global elimination program of measles.     

Characterisation of the RNA binding properties of the coronavirus infectious bronchitis virus nucleocapsid protein amino-terminal region

The coronavirus nucleocapsid (N) protein binds viral RNA to form the ribonucleocapsid and regulate RNA synthesis. The interaction of N protein with viral RNA was investigated using circular dichroism and surface plasmon resonance. N protein underwent a conformational change upon binding viral RNA and the data indicated electrostatic interactions were involved in the binding of the protein to RNA. Kinetic analysis suggested the amino-terminal region facilitates long-range non-specific interactions between N protein and viral RNA, thus bringing the RNA into close proximity to N protein allowing specific contacts to form via a 'lure' and 'lock' mechanism.     

A conformational epitope on the dimer of the fusion protein of respiratory syncytial virus detected in natural infections

A murine monoclonal antibody (MAb), 2D8, was used in immunofluorescence reactions to detect respiratory syncytial virus (RSV) antigen in clinical specimens. Nasopharyngeal epithelial cells from 63 of 66 children with RSV infections reacted with this MAb. The MAb was further characterized and was demonstrated to recognize a conformational epitope on the dimer of the fusion protein of RSV. No reaction was detected with the MAb, 2D8, on Western blots of antigen prepared from RSV-infected HEp-2 cells under reducing conditions. Under non-reducing conditions, 2D8 reacted with a 145-170 K protein; this reactivity was lost when the antigen preparation was heated to 100 degrees C. 2D8 reacted with purified F glycoprotein of RSV Long in an ELISA, neutralized infectivity of RSV by >50% at a dilution of 1:500, and was able to inhibit cell-to-cell fusion of RSV-infected cells. In a competitive ELISA, the epitope detected by 2D8 was localized to antigenic site A. The conformational epitope detected by 2D8 required protein dimerization and glycosylation for full reactivity. This report extends previous characterizations of the F protein in its native state in that the MAb defines a conformational epitope on the fusion protein dimer that is expressed in natural infections and elicits antibody that can neutralize virus infectivity and inhibit cell-to-cell fusion. In addition to its application as a diagnostic reagent, this MAb can be of use in testing preparations of RSV or purified F protein in which the purification or extraction processes could have destroyed conformational epitopes.     

A conformational switch in bacteriophage p22 portal protein primes genome injection

Double-stranded DNA (dsDNA) viruses such as herpesviruses and bacteriophages infect by delivering their genetic material into cells, a task mediated by a DNA channel called "portal protein." We have used electron cryomicroscopy to determine the structure of bacteriophage P22 portal protein in both the procapsid and mature capsid conformations. We find that, just as the viral capsid undergoes major conformational changes during virus maturation, the portal protein switches conformation from a procapsid to a mature phage state upon binding of gp4, the factor that initiates tail assembly. This dramatic conformational change traverses the entire length of the DNA channel, from the outside of the virus to the inner shell, and erects a large dome domain directly above the DNA channel that binds dsDNA inside the capsid. We hypothesize that this conformational change primes dsDNA for injection and directly couples completion of virus morphogenesis to a new cycle of infection.     

Structural difference recognized by a monoclonal antibody #404-11 between the rabies virus nucleocapsid (NC) produced in virus infected cells and the NC-like structures produced in the nucleoprotein (N) cDNA-transfected cells

We investigated structural changes in the rabies virus (HEP-Flury strain) nucleocapsid (NC) during the virus replication, for which we used two anti-nucleoprotein (N) monoclonal antibodies (mAbs), #404-11 (specific for a conformation-dependently exposed linear epitope) and #1-7-11 (specific for a conformational epitope which is exposed after the nucleocapsid formation). Both mAbs recognized the N protein of the viral NC, but not of the RNA-free N-P complex. The 1-7-11 and 404-11 epitopes could be mapped to the N-terminal and the C-terminal regions of N protein, respectively. Immunoprecipitation studies demonstrated that treatment of the NC either with the alkaline phosphatase or sodium deoxycholate (DOC) resulted in dissociation of most P proteins from the NC and in the reduced reactivity to mAb #404-11, but not to mAb #1-7-11. NC-like structures produced in the N cDNA-transfected cells displayed strong reactivity to mAb #1-7-11; however, reactivity to mAb #404-11 was very weak. And, coexpression with viral phosphoprotein (P) resulted in little increase in reactivity to mAb #404-11 of the NC-like structures, while the reactivity was significantly increased by cotransfection with P and the viral minigenome whose 3'- and 5'-end structures were derived from the viral genome. From these results, we assume that, although the 404-11 epitope is a linear one, the epitope-containing region is exposed only when N proteins encapsidate properly the viral RNA in collaboration with the P protein. Further, exposure of the 404-11 epitope region might be function-related, and be regulated by association and dissociation of the P protein.     

Steps in maturation of influenza A virus neuraminidase.

We have studied the maturation of the influenza A virus neuraminidase (NA), using monoclonal antibodies (MAbs) with different conformational specificities against the head domains of the N8 NA. The results obtained with radioimmunoprecipitation, together with previously published information, suggest the following steps in maturation of this molecule. First, the folding of the nascent NA leads to formation of the epitope recognized by MAb N8-10, a step that depends on the formation of intramolecular disulfide bonds. Second, monomers form dimers by an intermolecular disulfide linkage in the stalk, with a t1/2 of 2.5 min. Third, the epitope recognized by MAb N8-82 appears after dimerization, suggesting that oligomeric NAs may undergo conformational change with a t1/2 of 8 min. Finally, a tetramer-specific epitope recognized by MAb N8-4 appears on the NA with a t1/2 of 13 min. Epitope detection by MAb N8-4 was inhibited by tunicamycin treatment, suggesting that glycosylation of this molecule is required for proper tetramerization. Each of these proposed steps occurs in the endoplasmic reticulum of host cells, as demonstrated by treatment of virus-infected cells with brefeldin A or carbonyl cyanide m-chlorophenylhydrazine; subsequently, tetrameric NA is transported to the Golgi apparatus, where oligosaccharide processing is completed. Our findings also provide a possible explanation--lack of a functionally active conformation--for the absence of enzymatic function by NA monomers.     

Specific RNA binding by amino-terminal peptides of alfalfa mosaic virus coat protein.

Specific RNA-protein interactions and ribonucleoprotein complexes are essential for many biological processes, but our understanding of how ribonucleoprotein particles form and accomplish their biological functions is rudimentary. This paper describes the interaction of alfalfa mosaic virus (A1MV) coat protein or peptides with viral RNA. A1MV coat protein is necessary both for virus particle formation and for the initiation of replication of the three genomic RNAs. We have examined protein determinants required for specific RNA binding and analyzed potential structural changes elicited by complex formation. The results indicate that the amino-terminus of the viral coat protein, which lacks primary sequence homology with recognized RNA binding motifs, is both necessary and sufficient for binding to RNA. Circular dichroism spectra and electrophoretic mobility shift experiments suggest that the RNA conformation is altered when amino-terminal coat protein peptides bind to the viral RNA. The peptide--RNA interaction is functionally significant because the peptides will substitute for A1MV coat protein in initiating RNA replication. The apparent conformational change that accompanies RNA--peptide complex formation may generate a structure which, unlike the viral RNA alone, can be recognized by the viral replicase.     

Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies.

The objective of this study was to investigate how a conformational change in lipoprotein lipase (LPL) affects its molecular functions. Monoclonal antibodies (MAbs) were raised against purified bovine milk lipoprotein lipase. MAb 5D2 bound to human and bovine LPL both before and after denaturation of LPL. MAb 5F9 also recognized LPL from both species, but only after denaturation of the antigen, suggesting that a conformational change led to exposure of a previously hidden epitope. The MAbs were used in two sandwich enzyme-linked immunosorbent assays (ELISAs). One ELISA used the same MAb (5D2) to coat the plate and detect the bound antigen. This ELISA thus required the same epitope to be present in duplicate for detection (as would be the case with a dimeric antigen). The second ELISA used MAb 5F9 to coat the plate and MAb 5D2 to detect the antigen. This ELISA detected LPL only after it had been denatured. By measuring the same sample before and after denaturation with guanidine hydrochloride (GuHCl) in the 5F9 ELISA, and subtracting one from the other, a measure of native LPL was obtained. In inactivation experiments using human LPL, activity and the measure of LPL mass obtained in the 5D2 ELISA decreased and were related inversely to the measured mass obtained in the 5F9 ELISA which increased, indicating that loss of activity is closely linked to dimer dissociation and loss of native conformation. The effect of conformation and dimeric structure on LPL-heparin interaction was studied by heparin-Sepharose chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Matrix protein-specific IgA antibody inhibits measles virus replication by intracellular neutralization

Measles virus (MV) is still an imposing threat to public health. The matrix (M) protein has been shown not only to function as a structure block in the assembled MV virions, but also to regulate viral RNA synthesis, playing an important role in MV's replication and assembly. In the present study, we generated a panel of IgG monoclonal antibodies (MAbs) against M protein and successfully obtained one IgA MAb (5H7) from the IgG panel. Employing the polarized Vero cells grown in the two-chamber transwell model, we investigated whether M-specific 5H7 IgA MAb could suppress MV's replication and assembly. The data presented indicate that, while failing to show the activities of traditional neutralization and immune exclusion, M-specific IgA MAb was able to effectively inhibit viral replication by intracellular neutralization (78%), supporting the notion that the M protein is important for MV assembly and replication and implying that the M protein was an effective target antigen. The data also showed that MV had a long entry and assembly phase during viral replication, providing an extended window for IgA intervention. The colocalization of M proteins and M-specific 5H7 IgA MAbs demonstrated that the intracellular neutralization was due to the direct binding of the M-specific 5H7 IgA MAbs to the M proteins. In summary, the present study has added another example showing that IgA antibodies targeting internal viral antigens could proactively participate in mucosal immune protection by intracellular neutralization and has provided evidence that M protein might be included as a target antigen in future MV vaccine design.     

Expression of Measles Virus V Protein Is Associated with Pathogenicity and Control of Viral RNA Synthesis

Nonstructural proteins encoded by measles virus (MV) include the V protein which is translated from an edited P mRNA. V protein is not associated with intracellular or released viral particles and has recently been found to be dispensable for MV propagation in cell culture (H. Schneider, K. Kaelin, and M. A. Billeter, Virology 227:314–322, 1997). Using recombinant MVs (strain Edmonston [ED]) genetically engineered to overexpress V protein (ED-V+) or to be deficient for V protein (ED-V−), we found that in the absence of V both MV-specific proteins and RNAs accumulated to levels higher than those in the parental MV molecular clone (ED-tag), whereas MV-specific gene expression was strongly attenuated in human U-87 glioblastomas cells after infection with ED-V+. The titers of virus released from these cells 48 h after infection with either V mutant virus were lower than those from cells infected with ED-tag. Similarly, significantly reduced titers of infectious virus were reisolated from lung tissue of cotton rats (Sigmodon hispidus) after intranasal infection with both editing mutants compared to titers isolated from ED-tag-infected animals. In cell culture, expression of V protein led to a redistribution of MV N protein in doubly transfected Cos-7 cells, indicating that these proteins form heterologous complexes. This interaction was further confirmed by using a two-hybrid approach with both proteins expressed as Gal4 or VP16 fusion products. Moreover, V protein efficiently competed complexes formed between MV N and P proteins. These findings indicate that V protein acts to balance accumulation of viral gene products in cell culture, and this may be dependent on its interaction with MV N protein. Furthermore, expression of V protein may contribute to viral pathogenicity in vivo.     

Interaction of measles virus (Hallé strain) with CD46: evidence that a common binding site on CD46 facilitates both CD46 downregulation and MV infection.

CD46 acts as a cellular receptor for vaccine strains of measles virus (MV). The MV/CD46 interaction-mediated by the MV attachment glycoprotein, the hemagglutinin (H)-not only facilitates infection but also induces CD46 downregulation. A conflict of opinion exists as to whether a single MVH binding site on CD46, or two separate sites, facilitates the two phenomena. To investigate this conundrum we first tested and compared a panel of CD46-specific monoclonal antibodies (mAbs) for their capacity to block both processes. One (mAb 13/42) abrogated both MV fusion and CD46 downregulation. Mutation of an amino acid (arg59 in the SCR1 of CD46) essential for the epitope of mAb 13/42 resulted in the abrogation of both CD46 downregulation and viral fusion. This strongly suggests that the same MV binding site on CD46 is responsible for both CD46 downregulation and MV infection.     

The hantavirus nucleocapsid protein recognizes specific features of the viral RNA panhandle and is altered in conformation upon RNA binding

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is the principal structural component of the viral capsid. N forms a stable trimer that specifically recognizes the panhandle structure formed by the viral RNA termini. We used trimeric glutathione S-transferase (GST)-N protein and small RNA panhandles to examine the requirements for specific recognition by Sin Nombre hantavirus N. Trimeric GST-N recognizes the panhandles of the three viral RNAs (S, M, and L) with high affinity, whereas the corresponding plus-strand panhandles of the complementary RNA are recognized with lower affinity. Based on analysis of nucleotide substitutions that alter either the higher-order structure of the panhandle or the primary sequence of the panhandle, both secondary structure and primary sequence are necessary for stable interaction with N. A panhandle 23 nucleotides long is necessary and sufficient for high-affinity binding by N, and stoichiometry calculations indicate that a single N trimer interacts with a single panhandle. Surprisingly, displacement of the panhandle structure away from the terminus does not eliminate recognition by N. The binding of N to the panhandle is an entropy-driven process resulting in initial stable N-RNA interaction followed by a conformational change in N. Taken together, these data provide insight into the molecular events that take place during interaction of N with the panhandle and suggest that specific high-affinity interaction between an RNA binding domain of trimeric N and the panhandle is required for encapsidation of the three viral RNAs.     

Hydrogen bonding at a conserved threonine in lentivirus capsid is required for virus replication

The N terminus of the capsid protein (CA) undergoes a considerable conformational change when the human immunodeficiency virus (HIV) protease cleaves it free from the Pr55(Gag) polyprotein. This rearrangement is thought to facilitate the establishment of specific CA-CA interactions that are required for the formation of the mature viral core. Substitution of amino acids that are critical for this refolding of the N terminus is generally detrimental to virus replication and mature virion core morphology. Here, we identify a conserved threonine in simian immunodeficiency virus (SIV) CA, T(47)(CA), that is requisite for viral replication. Replacement of T(47)(CA) in the infectious viral clone SIVmac239 with amino acids with different hydrogen-bonding capabilities and analysis of the effects of these substitutions at key steps in the viral life cycle demonstrate that hydrogen bonding at this position is important for virus infectivity and virion release. In the HIV-based homology model of the mature SIV CA N terminus presented in this study, T(47)(CA) forms several hydrogen bonds with a proximal aspartate, D(50)(CA). This model, coupled with strong phenotypic similarities between viral substitution mutants of each of these two residues in all of the virological assays described herein, indicates that hydrogen bonding between T(47)(CA) and D(50)(CA) is likely required for viral replication. As hydrogen bonding between these two residues is present in HIV CA as well, this interaction presents a potential target for antiviral drug design.     

Monoclonal Antibodies Recognize Conformational Epitopes on Wild-type and Recombinant Mutant Amidases from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Hybridoma technology was used to raise monoclonal antibodies (MAbs) against wild-type amidase from Pseudomonas aeruginosa. Hybridoma clones secreting polyol-responsive MAbs (PR-MAbs) were screened that bind antigen tightly. but release under mild- and non-denaturing elution conditions, which can be used as ligands in immunoaffinity chromatography. Two of these hybridoma clones (C9E4 and B1E4) secreting MAbs against wild-type amidase were selected in order to check if they are PR-MAbs by using ELISA-elution assay. These hybridoma cell lines secreted MAbs of IgG class which were purified in a single step by Protein A-Sepharose CL-4B chromatography, which revealed two protein bands on SDS-PAGE. Specificity studies of MAb C9E4 revealed that it recognized a common epitope on wild-type and mutant T103I amidases as determined by direct ELISA, as well as by Western blotting under native conditions. This MAb exhibited a higher-affinity constant (K) for the mutant T103I amidase than for the wild-type enzyme. However, this MAb did not recognize either wild-type or mutant T103I enzymes under denaturing conditions suggesting that it binds to a conformation-sensitive epitope on amidase molecule. On the other hand, it also does not recognize either native or denatured forms of mutant C91A amidase suggesting that this substitution disrupted the conformational epitope present on amidase molecule. Furthermore, MAb C9E4 inhibited about 80% of wild-type amidase activity, whereas it activated about 80% of mutant amidase (T103I) activity. However, this MAb did not affect mutant C91A amidase activity which is in agreement with other results presented in this work. The data presented in this work suggest that this MAb acts as a powerful probe to detect conformational changes in native and denatured amidases as well as to differentiate wild-type and mutant (T103I and C91A) amidases.     

Measles virus targets DC-SIGN to enhance dendritic cell infection

Dendritic cells (DCs) are involved in the pathogenesis of measles virus (MV) infection by inducing immune suppression and possibly spreading the virus from the respiratory tract to lymphatic tissues. It is becoming evident that DC function can be modulated by the involvement of different receptors in pathogen interaction. Therefore, we have investigated the relative contributions of different MV-specific receptors on DCs to MV uptake into and infection of these cells. DCs express the MV receptors CD46 and CD150, and we demonstrate that the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is a novel receptor for laboratory-adapted and wild-type MV strains. The ligands for DC-SIGN are both MV glycoproteins F and H. In contrast to CD46 and CD150, DC-SIGN does not support MV entry, since DC-SIGN does not confer susceptibility when stably expressed in CHO cells. However, DC-SIGN is important for the infection of immature DCs with MV, since both attachment and infection of immature DCs with MV are blocked in the presence of DC-SIGN inhibitors. Our data demonstrate that DC-SIGN is crucial as an attachment receptor to enhance CD46/CD150-mediated infection of DCs in cis. Moreover, MV might not only target DC-SIGN to infect DCs but may also use DC-SIGN for viral transmission and immune suppression.     

Conformational epitope mapping of OmpC, a major cell surface antigen from Salmonella typhi

The outer membrane protein OmpC, a trimer made of 16 stranded beta-barrel monomers, is a major cell surface antigen from the human pathogen Salmonella typhi. The relative stability of the epitopes recognising a Salmonella specific MAb (referred as MPN5) and an Enterobacteria specific MAb (referred as P7D8) and the role of the trimeric organisation has been probed using gel electrophoresis and monoclonal antibodies. The assembly of the trimer and the stability of the beta-barrel are found to be important for epitope presentation. The Salmonella specific conformational epitope is found to be more stable than the Enterobacteria specific one. The important residues of the Salmonella specific (Asp 25 of loop 1, Asp 340 of loop 8, Lys 334 of loop 8, and Tyr 210 of loop 5) and the Enterobacteria specific (Asp 25 of loop 1, Tyr 210 of loop 5, and Lys 152 of loop 4) conformational epitope have been identified using monoclonal antibodies, chemical modification, and solid phase binding methods.