Genetic and biochemical studies on the assembly of an enveloped virus.
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Alphavirus↗
Biomedical subjects
R J Kuhn
Publications and source records attributed to R J Kuhn.
Interaction of coxsackievirus B3 with the full length coxsackievirus-adenovirus receptor.
Group B coxsackieviruses (CVB) utilize the coxsackievirus-adenovirus receptor (CAR) to recognize host cells. CAR is a membrane protein with two Ig-like extracellular domains (D1 and D2), a transmembrane domain and a cytoplasmic domain. The three-dimensional structure of coxsackievirus B3 (CVB3) in complex with full length human CAR and also with the D1D2 fragment of CAR were determined to approximately 22 A resolution using cryo-electron microscopy (cryo-EM). Pairs of transmembrane domains of CAR associate with each other in a detergent cloud that mimics a cellular plasma membrane. This is the first view of a virus-receptor interaction at this resolution that includes the transmembrane and cytoplasmic portion of the receptor. CAR binds with the distal end of domain D1 in the canyon of CVB3, similar to how other receptor molecules bind to entero- and rhinoviruses. The previously described interface of CAR with the adenovirus knob protein utilizes a side surface of D1.
Adenoviridae↗
Alphavirus nucleocapsid protein contains a putative coiled coil alpha-helix important for core assembly.
The alphavirus nucleocapsid core is formed through the energetic contributions of multiple noncovalent interactions mediated by the capsid protein. This protein consists of a poorly conserved N-terminal region of unknown function and a C-terminal conserved autoprotease domain with a major role in virion formation. In this study, an 18-amino-acid conserved region, predicted to fold into an alpha-helix (helix I) and embedded in a low-complexity sequence enriched with basic and Pro residues, has been identified in the N-terminal region of the alphavirus capsid proteins. In Sindbis virus, helix I spans residues 38 to 55 and contains three conserved leucine residues, L38, L45, and L52, conforming to the heptad amino acid organization evident in leucine zipper proteins. Helix I consists of an N-terminally truncated heptad and two complete heptad repeats with beta-branched residues and conserved leucine residues occupying the a and d positions of the helix, respectively. Complete or partial deletion of helix I, or single-site substitutions at the conserved leucine residues (L45 and L52), caused a significant decrease in virus replication. The mutant viruses were more sensitive to elevated temperature than wild-type virus. These mutant viruses also failed to accumulate cores in the cytoplasm of infected cells, although they did not have defects in protein translation or processing. Analysis of these mutants using an in vitro assembly system indicated that the majority were defective in core particle assembly. Furthermore, mutant proteins showed a trans-dominant negative phenotype in in vitro assembly reactions involving mutant and wild-type proteins. We propose that helix I plays a central role in the assembly of nucleocapsid cores through coiled coil interactions. These interactions may stabilize subviral intermediates formed through the interactions of the C-terminal domain of the capsid protein and the genomic RNA and contribute to the stability of the virion.
Alphavirus↗
An amino acid substitution in the coding region of the E2 glycoprotein adapts Ross River virus to utilize heparan sulfate as an attachment moiety.
Passage of Ross River virus strain NB5092 in avian cells has been previously shown to select for virus variants that have enhanced replication in these cells. Sequencing of these variants identified two independent sites that might be responsible for the phenotype. We now demonstrate, using a molecular cDNA clone of the wild-type T48 strain, that an amino acid substitution at residue 218 in the E2 glycoprotein can account for the phenotype. Substitutions that replaced the wild-type asparagine with basic residues had enhanced replication in avian cells while acidic or neutral residues had little or no observable effect. Ross River virus mutants that had increased replication in avian cells also grew better in BHK cells than the wild-type virus, whereas the remaining mutants were unaffected in growth. Replication in both BHK and avian cells of Ross River virus mutants N218K and N218R was inhibited by the presence of heparin or by the pretreatment of the cells with heparinase. Binding of the mutants, but not of the wild type, to a heparin-Sepharose column produced binding comparable to that of Sindbis virus, which has previously been shown to bind heparin. Replication of these mutants was also adversely affected when they were grown in a CHO cell line that was deficient in heparan sulfate production. These results demonstrate that amino acid 218 of the E2 glycoprotein can be modified to create an heparan sulfate binding site and this modification expands the host range of Ross River virus in cultured cells to cells of avian origin.
Amino Acid Substitution↗
Interaction of coxsackievirus A21 with its cellular receptor, ICAM-1.
Coxsackievirus A21 (CAV21), like human rhinoviruses (HRVs), is a causative agent of the common cold. It uses the same cellular receptor, intercellular adhesion molecule 1 (ICAM-1), as does the major group of HRVs; unlike HRVs, however, it is stable at acid pH. The cryoelectron microscopy (cryoEM) image reconstruction of CAV21 is consistent with the highly homologous crystal structure of poliovirus 1; like other enteroviruses and HRVs, CAV21 has a canyon-like depression around each of the 12 fivefold vertices. A cryoEM reconstruction of CAV21 complexed with ICAM-1 shows all five domains of the extracellular component of ICAM-1. The known atomic structure of the ICAM-1 amino-terminal domains D1 and D2 has been fitted into the cryoEM density of the complex. The site of ICAM-1 binding within the canyon of CAV21 overlaps the site of receptor recognition utilized by rhinoviruses and polioviruses. Interactions within this common region may be essential for triggering viral destabilization after attachment to susceptible cells.
Amino Acid Sequence↗
Ross River virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production.
Pseudotyped retroviruses have important applications as vectors for gene transfer and gene therapy and as tools for the study of viral glycoprotein function. Recombinant Moloney murine leukemia virus (Mo-MuLV)-based retrovirus particles efficiently incorporate the glycoproteins of the alphavirus Ross River virus (RRV) and utilize them for entry into cells. Stable cell lines that produce the RRV glycoprotein-pseudotyped retroviruses for prolonged periods of time have been constructed. The pseudotyped viruses have a broadened host range, can be concentrated to high titer, and mediate stable transduction of genes into cells. The RRV glycoprotein-pseudotyped retroviruses and the cells that produce them have been employed to demonstrate that RRV glycoprotein-mediated viral entry occurs through endocytosis and that membrane fusion requires acidic pH. Alphavirus glycoprotein-pseudotyped retroviruses have significant advantages as reagents for the study of the biochemistry and prevention of alphavirus entry and as preferred vectors for stable gene transfer and gene therapy protocols.
3T3 Cells↗
In vitro assembly of Sindbis virus core-like particles from cross-linked dimers of truncated and mutant capsid proteins.
A nucleic acid-bound capsid protein dimer was previously identified using a Sindbis virus in vitro nucleocapsid assembly system and cross-linking reagents. Cross-link mapping, in combination with a model of the nucleocapsid core, suggested that this dimer contained one monomer from each of two adjacent capsomeres. This intercapsomere dimer is believed to be the initial intermediate in the nucleocapsid core assembly mechanism. This paper presents the purification of cross-linked dimers of a truncated capsid protein and the partial purification of cross-linked dimers of a full-length assembly-defective mutant. The assembly of core-like particles from these cross-linked capsid protein dimers is demonstrated. Core-like particles generated from cross-linked full-length mutant CP(19-264)L52D were examined by electron microscopy and appeared to have a morphology similar to that of wild-type in vitro-assembled core-like particles, although a slight size difference was often visible. Truncated cross-linked CP(81-264) dimers generated core-like particles as well. These core-like particles could subsequently be disassembled when reversible cross-linking reagents were used to form the dimers. The ability of the covalent intercapsomere cross-link to rescue capsid proteins with assembly defects or truncations in the amino-terminal region of the capsid protein supports the previous model of assembly and suggests a possible role for the amino-terminal region of the protein.
Blotting, Western↗
Formulation of aerosolized therapeutics.
Pseudomonas aeruginosa (PA), which colonizes the airways of approximately 90% of patients with cystic fibrosis (CF) at some point during their lives, is an important contributor to the vicious cycle of infection and inflammation leading to bronchiectasis and eventual respiratory failure. Oral antibiotic therapy is often ineffective in treating PA infections. Instead, in-hospital IV aminoglycoside therapy administered in combination with other IV antibiotics, such as beta-lactams or quinolones, is the mainstay of treatment. The specific chemical and physical properties of CF sputum require high serum antibiotic levels for effective antimicrobial treatment; however, IV aminoglycoside therapy is associated with an increased risk of ototoxicity and nephrotoxicity. In an attempt to avoid systemic toxicity and effectively treat PA infections, clinicians have combined IV antibiotics with sterile solutions of saline or water to aerosolize the mixture for inhalation. Experience with such "home brews" has clearly demonstrated that IV preparations are neither intended nor medically indicated for inhalation. Patients may experience coughing, mucosal irritation, or bronchospasm in response to the preservatives, stabilizing agents, and other additives commonly found in IV preparations. While the rationale for aerosolized drug delivery remains compelling, concerns about uniform dose delivery, ineffective nebulization, and therapeutic adherence arise. Since the 1940s, when these efforts began, ongoing research and clinical trials have identified several additional factors affecting inhaled drug delivery and deposition in the airways. This article chronicles some of the challenges faced by researchers and elucidates factors critical to the reformulation of a safe and effective antibiotic solution for aerosolized delivery.
Aerosols↗
Susceptibility of pseudomonas aeruginosa to cefepime versus ceftazidime in patients with cystic fibrosis.
STUDY OBJECTIVES: To compare the susceptibility of respiratory cultures of Pseudomonas aeruginosa obtained from patients with cystic fibrosis to cefepime versus ceftazidime. The pattern of cumulative resistance of P aeruginosa to cefepime in patients who had received at least one treatment course of cefepime between two sputum cultures was also characterized. DESIGN: Prospective consecutive data collection. SETTING: University-affiliated cystic fibrosis clinic and medical center. PATIENTS: Eighty patients with cystic fibrosis who had at least one sputum culture positive for P aeruginosa with reported microbiologic susceptibilities to cefepime and ceftazidime. INTERVENTION: Patient data was collected and analyzed. Measurements and Main Results. Two hundred and thirty-one P aeruginosa isolates were collected over 6 months. A total of 16.4% and 8.7% of the isolates were nonsusceptible to cefepime and ceftazidime, respectively (p=0.01). In eight patients who had not received cefepime before the study period, nonsusceptibility was 11.8% and 27.2% before and after exposure to cefepime, respectively. CONCLUSIONS: Susceptibility of P. aeruginosa isolates in patients with cystic fibrosis was lower with cefepime than with ceftazidime. Follow-up surveillance to determine changes in susceptibility of P aeruginosa isolates to cefepime is warranted.
Adolescent↗
Active transport of nitrofurantoin into human milk.
STUDY OBJECTIVE: To determine the extent to which nitrofurantoin is transferred into human milk. DESIGN: Prospective, single-dose pharmacokinetic study. SETTING: University-affiliated clinical research center. PATIENTS: Four healthy lactating women 8-26 weeks postpartum. INTERVENTION: All subjects received a single, oral, 100-mg dose of nitrofurantoin macrocrystals with food. Serial serum and milk samples were obtained and analyzed by high-performance liquid chromatography. MEASUREMENTS AND MAIN RESULTS: Milk pH, milk fat partitioning, and protein binding in serum and milk were determined. Predicted milk:serum ratio (M:S) was compared with the observed M:S. Nitrofurantoin M:S predicted was 0.28+/-0.05, whereas M:S observed was 6.21+/-2.71. Average milk concentration was 1.3 mg/L, and estimated suckling infant dosage was 0.2 mg/kg/day or 6% of maternal dose (mg/kg). CONCLUSIONS: Nitrofurantoin is actively transported into human milk, achieving concentrations in milk greatly exceeding those in serum. Concern is warranted for suckling infants younger than 1 month old, or for infants with a high frequency of glucose-6-phosphate dehydrogenase deficiency or sensitivity to nitrofurantoin.
Administration, Oral↗
Will the polio niche remain vacant?
C-Cluster enteroviruses (C-CEVs), consisting of Coxsackie A viruses (C-CAV1, 11, 13, 15, 17, 18, 19, 20, 21, 22, 24, 24v) and polioviruses (PV1, 2, 3), have been grouped together in relation to their genomic sequences. On the basis of disease syndromes caused in humans, however, C-CAVs and PVs are vastly different: the former cause respiratory disease, just like the major receptor group rhinoviruses (magHRV), whereas PVs, on invasion of the CNS, can cause poliomyelitis. It is assumed that the difference in pathogenesis of C-CEVs is governed predominantly by cellular receptor specificity. C-CAVs use ICAM-1, just like magHRV, whereas PVs uniquely use CD155. Both ICAM-1 and CD155 are Ig-like molecules. Remarkably, based on a phylogenetic analysis of non-structural proteins, CAV 11, 13, 17 and 18 are interleaved with, rather than separated from, the three PV serotypes, e.g. PV1 is more closely related to CAV18 that to PV2. This observation suggests that PVs may have emerged from a pool of C-CAVs by evolving a unique receptor specificity. We have been studying virion structure, virion/receptor interactions, genetics, and the molecular biology of C-CEVs with the objective of identifying the molecular basis of phenotypic diversity of these viruses. Of particular interest is the prospect that C-CEVs can be genetically manipulated to switch their receptor affinity: from CD155 to ICAM-1 for PVs, or from ICAM-1 to CD155 for C-CAVs. We propose a hypothesis that in a world free of poliovirus and anti-poliovirus neutralizing antibodies C-CAVs would be given a greater chance to switch receptor specificity from ICAM-1 to CD155 and thus, to evolve gradually into a new polio-like virus.
Animals↗
Cell recognition and entry by rhino- and enteroviruses.
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Animals↗
Interaction of the poliovirus receptor with poliovirus.
The structure of the extracellular, three-domain poliovirus receptor (CD155) complexed with poliovirus (serotype 1) has been determined to 22-A resolution by means of cryo-electron microscopy and three-dimensional image-reconstruction techniques. Density corresponding to the receptor was isolated in a difference electron density map and fitted with known structures, homologous to those of the three individual CD155 Ig-like domains. The fit was confirmed by the location of carbohydrate moieties in the CD155 glycoprotein, the conserved properties of elbow angles in the structures of cell surface molecules with Ig-like folds, and the concordance with prior results of CD155 and poliovirus mutagenesis. CD155 binds in the poliovirus "canyon" and has a footprint similar to that of the intercellular adhesion molecule-1 receptor on human rhinoviruses. However, the orientation of the long, slender CD155 molecule relative to the poliovirus surface is quite different from the orientation of intercellular adhesion molecule-1 on rhinoviruses. In addition, the residues that provide specificity of recognition differ for the two receptors. The principal feature of receptor binding common to these two picornaviruses is the site in the canyon at which binding occurs. This site may be a trigger for initiation of the subsequent uncoating step required for viral infection.
Amino Acid Sequence↗
Nucleic acid-dependent cross-linking of the nucleocapsid protein of Sindbis virus.
The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway.
Capsid↗
Photobiological properties of positively charged methylene violet analogs.
O-Methyl methylene violet (OMeMV), O-methyl bromomethylene violet (OMeBrMV) and O-methyl iodomethylene violet (OMeIMV) have been prepared in order to test their potential utility as anti-viral and anti-tumor phototoxic dyes. Rates of photosensitized toxicity of KB cells with 633 nm irradiation are (x 10(-19) photon-1): 2.4, 2.2, 1.9 and 0.17 for OMeIMV, OMeBrMV, methylene violet (MV) and OMeMV, respectively. Rates of photosensitized inactivation of Sindbis virus in phosphate-buffered saline with 633 nm irradiation are (x 10(-18) photon-1): 3.3, 1.8, 0.99, 0.15 for MV, OMeIMV, OMeBrMV and OMeMV, respectively. Quantum efficiencies for singlet oxygen formation were determined as OMeIMV, 0.64; OMeBrMV, 0.40; OMeMV, 0.054. Titration of the dyes with double-stranded (ds)DNA resulted in bathochromic shifts and hypochromic effects in the visible absorption spectra. Association constants for interaction of the methylated dyes with dsDNA of approximately 1 x 10(5) M-1 were determined by Scatchard analysis of equilibrium dialysis and UV absorption titration data. Photolysis of the halogenated dyes with DNA under argon led to covalent bond formation with the nucleic acid; there was no evidence of covalent binding in the dark.
Antiviral Agents↗
In vitro assembly of alphavirus cores by using nucleocapsid protein expressed in Escherichia coli.
The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19-264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19-264) or could inhibit assembly if its molar ratio relative to CP(19-264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly.
Base Sequence↗
Probing the potential glycoprotein binding site of sindbis virus capsid protein with dioxane and model building.
Alphavirus budding from the plasma membrane is initiated by the specific interaction of the nucleocapsid with the cytoplasmic domain of the glycoprotein E2. It was proposed (Lee et al., Structure 4:531-541, 1996) that binding of the capsid protein residues 108 to 110 (the "N-terminal arm" residues) to a hydrophobic pocket on the surface of the neighboring capsid protein in the crystal structure mimics the binding of the E2 C-terminal residues into this pocket. In addition, structural comparisons of wild-type and mutant Sindbis virus capsid protein (SCP) and Semliki Forest virus capsid protein suggested that budding is associated with a switch between two conformations of the hydrophobic pocket. To test the proposed mechanism, SCP(114-264), which is missing the N-terminal arm, was crystallized to examine the pocket conformation when the pocket is empty. However, the pocket was occupied by dioxane molecules from the crystallization solution. The pocket conformation was the same as that when it was occupied by the N-terminal arm, demonstrating that the pocket favors binding ligands of appropriate size and shape.
Amino Acid Sequence↗
Alphavirus budding is dependent on the interaction between the nucleocapsid and hydrophobic amino acids on the cytoplasmic domain of the E2 envelope glycoprotein.
The interaction between the nucleocapsid core and the glycoprotein spikes is a critical component in the budding process of alphaviruses. A molecular model was previously proposed which suggested that this interaction was mediated by the binding of the cytoplasmic domain of glycoprotein E2 into a hydrophobic pocket found on the surface of the nucleocapsid protein [S. Lee, K. E. Owen, H.-K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. Rossmann, and R. J. Kuhn (1996) Structure 4, 531-541; U. Skoging, M. Vihinen, L. Nilsson, and P. Liljeström (1996) Structure 4, 519-529]. Two hydrophobic amino acids in the cytoplasmic domain of E2 were predicted to be important in the contact between the proteins. One of the residues, Y400 (Sindbis virus numbering), had previously been shown by mutational studies to be important in the budding of Semliki Forest virus [H. Zhao, B. Lindqvist, H. Garoff, C. H. von Bonsdorf, and P. Liljeström (1994) EMBO J. 13, 4204-4211]. The role of the second residue, L402, had not been examined. By creating a panel of amino acid substitutions at this residue, followed by phenotypic analysis of rescued mutant viruses, we now show that L402 is critical for the production of Sindbis virus. Substitutions at this amino acid inhibit budding, and the data suggest the L402 plays an important role in the interaction, between the glycoprotein and the nucleocapsid core. These data support the model and suggest that the proposed molecular interactions are important for the budding of alphaviruses from the cell.
Amino Acids↗