Characterizing and controlling the inherent dynamics of cyclophilin-A

With the recent advances in NMR relaxation techniques, protein motions on functionally important timescales can be studied at atomic resolution. Here, we have used NMR-based relaxation experiments at several temperatures and both 600 and 900 MHz to characterize the inherent dynamics of the enzyme cyclophilin-A (CypA). We have discovered multiple chemical exchange processes within the enzyme that form a “dynamic continuum” that spans 20–30 Å comprising active site residues and residues proximal to the active site. By combining mutagenesis with these NMR relaxation techniques, a simple method of counting the dynamically sampled conformations has been developed. Surprisingly, a combination of point mutations has allowed for the specific regulation of many of the exchange processes that occur within CypA, suggesting that the dynamics of an enzyme may be engineered.     

Residues in the Heptad Repeat A Region of the Fusion Protein Modulate the Virulence of Sendai Virus in Mice

While the molecular basis of fusion (F) protein refolding during membrane fusion has been studied extensively in vitro, little is known about the biological significance of membrane fusion activity in parainfluenza virus replication and pathogenesis in vivo. Two recombinant Sendai viruses, F-L179V and F-K180Q, were generated that contain F protein mutations in the heptad repeat A region of the ectodomain, a region of the protein known to regulate F protein activation. In vitro, the F-L179V virus caused increased syncytium formation (cell-cell membrane fusion) yet had a rate of replication and levels of F protein expression and cleavage similar to wild-type virus. The F-K180Q virus had a reduced replication rate along with reduced levels of F protein expression, cleavage, and fusogenicity. In DBA/2 mice, the hyperfusogenic F-L179V virus induced greater morbidity and mortality than wild-type virus, while the attenuated F-K180Q virus was much less pathogenic. During the first week of infection, virus replication and inflammation in the lungs were similar for wild-type and F-L179V viruses. After approximately 1 week of infection, the clearance of F-L179V virus was delayed, and more extensive interstitial inflammation and necrosis were observed in the lungs, affecting entire lobes of the lungs and having significantly greater numbers of syncytial cell masses in alveolar spaces on day 10. On the other hand, the slower-growing F-K180Q virus caused much less extensive inflammation than wild-type virus, presumably due to its reduced replication rate, and did not cause observable syncytium formation in the lungs. Overall, the results show that residues in the heptad repeat A region of the F protein modulate the virulence of Sendai virus in mice by influencing both the spread and clearance of the virus and the extent and severity of inflammation. An understanding of how the F protein contributes to infection and inflammation in vivo may assist in the development of antiviral therapies against respiratory paramyxoviruses.Sendai virus (SeV), a murine parainfluenza virus (PIV), belongs to the genus Respirovirus within the family Paramyxoviridae (33). Sendai virus is the murine counterpart of human parainfluenza virus 1 (HPIV1), and these two viruses share high sequence homology and antigenic cross-reactivity (23, 38, 58). Both Sendai virus and HPIV1 cause respiratory diseases in their hosts that range from mild to severe, with the greatest morbidity and mortality occurring in immunocompromised hosts (3, 17). In pediatric medicine, HPIV1 is an important cause of bronchiolitis, pneumonia, and laryngotracheobronchitis, or croup (11). Other members of the genus Respirovirus include human and bovine forms of PIV3 (30).Like other paramyxoviruses, Sendai virus is an enveloped, nonsegmented, negative-strand RNA virus that invades host cells by fusion (F) protein-mediated membrane fusion at the plasma membrane (33). The receptor binding protein for Sendai virus, as well as the other parainfluenza viruses, is the hemagglutinin-neuraminidase (HN) protein. During viral entry, the HN protein binds sialic acid-containing receptors on the surfaces of host cells and then triggers the F protein to refold and cause membrane fusion (34, 40). Paramyxovirus replication occurs in the cytoplasm of infected cells, where the viral nucleocapsid is formed by the encapsidation of the viral genome with the viral nucleoprotein (N), phosphoprotein (P), and the large RNA-dependent RNA-polymerase (L) protein (33). The assembly and budding of infectious parainfluenza virions from the plasma membrane are mediated largely by the matrix (M) protein, which interacts with the viral nucleocapsid and the cytoplasmic tails of the HN and F proteins (56, 63).The paramyxovirus F protein mediates both virus-cell fusion and cell-cell fusion. Similar to other class I viral fusion proteins, paramyxovirus F proteins are expressed on the surfaces of infected cells and virions as trimers that are trapped in metastable (high energy) conformations (29, 54, 71, 73). In order to become activated for membrane fusion, uncleaved F₀ precursor protein trimers must be cleaved into a fusion-capable complex formed by F₁ and F₂ subunits (55). Field isolates of Sendai virus that have a monobasic cleavage site are cleavage activated by tryptase Clara secreted from respiratory epithelial cells (32, 69) while the pantropic F1-R laboratory isolate of Sendai virus has a mutated cleavage site and is cleaved by more ubiquitously expressed proteases (41, 67). Paramyxovirus F proteins have several regions involved in F protein conformational changes during membrane fusion: a hydrophobic fusion peptide, two 4-3 heptad repeat regions (designated heptad repeat A [HRA] and HRB), a transmembrane domain, and a cytoplasmic tail. The prefusion form of the PIV5 F₀ protein has a mushroom-like shape formed by a large globular head attached to a rod-like stalk formed by the HRB region (76). Upon triggering by the HN protein, the HRB region dissociates, the HRA region springs into a coiled coil, and the fusion peptide is inserted into the target membrane (52). Membrane fusion is catalyzed by the formation of a coiled-coil hairpin structure (2, 7, 75, 78), formed by the HRA and HRB regions, that juxtaposes the membrane-interacting fusion peptide and transmembrane domains (52). We recently performed a mutational analysis on a 10-residue sequence in the HRA region of the Sendai virus F protein (37) that forms a β-strand-turn-α-helix structure in the prefusion conformation and part of a triple-stranded coiled coil in the hairpin conformation (75, 76). The mutated residues were found to play important roles in regulating the activation and membrane fusion activity of the Sendai virus F protein, showing that F protein refolding is regulated by residues that undergo dramatic changes in secondary and tertiary structure between the prefusion and hairpin conformations.Upon triggering by the HN protein, cell surface-expressed F protein trimers mediate cell-cell fusion (syncytium formation) and extend infection in a local area (55). In nonpolarized epithelial cells, virus-induced syncytium formation has long been considered a hallmark of in vitro cytopathogenesis by respiratory paramyxoviruses. However, many questions remain regarding the extent of envelope glycoprotein expression, parainfluenza virus budding, and syncytium formation at the basolateral surfaces of polarized cells (4, 77). In an in vitro model of human airway epithelium (HAE), HPIV3 has been shown to infect ciliated epithelial cells exclusively, predominantly at the apical surface, causing little virus-mediated cytopathology, no spread of the virus beyond ciliated cells, and no syncytium formation (77). As the HAE model lacks innate and adaptive immune cells, this model would not reveal the formation of syncytia involving all cell types in the respiratory tract that are present during infection, including those that play a role in the host response to infection. In immunocompetent mice, the replication of field isolates of Sendai virus is restricted to the respiratory tract, and progeny virions bud from the apical surfaces of polarized epithelial cells (68). While syncytial cell formation in the bronchiolar epithelia of mice infected with Sendai virus has been reported previously (28), the timing of giant cell formation and its contribution to the spread of the virus and the disease it induces in the respiratory tract remain unknown.To test the hypothesis that the fusogenicity of the F protein contributes to the pathogenicity of Sendai virus in mice, the natural host of this virus, we generated two recombinant Sendai viruses containing F protein mutations in the heptad repeat A region that were found previously to either increase or decrease its fusogenic activity when the F protein was expressed from plasmid DNA constructs (37). In the present study, the effects of the F protein mutations on virus replication, F protein expression, F protein cleavage, and syncytium formation were characterized in vitro. The hyperfusogenic F-L179V virus was found to induce greater morbidity and mortality in DBA/2 mice than wild-type virus, whereas the hypofusogenic and attenuated F-K180Q virus was found to be much less pathogenic. After 1 week of infection, the F-L179V virus induced more extensive interstitial inflammation and necrosis in the lungs than the wild-type virus, including a greater number of syncytial cell masses. On the other hand, the attenuated F-K180Q virus caused much less extensive inflammation than wild-type virus and did not cause observable syncytium formation in the lungs. A comparison of 50% minimal lethal dose (MLD₅₀) values, lung titers, and histopathologic changes reveals a correlation between the membrane fusion activity of the F protein and the virulence of Sendai virus in mice.     

Expression, purification, and biochemical characterization of a recombinant Iectin of Sarcocystis muris (Apicomplexa) cyst merozoites

The mature major microneme protein of Sarcocystis muris cyst merozoites, which is known as a dimeric lectin with high affinity to galactose and some of its derivatives, was expressed in Escherichia coli as a histidine-tagged fusion protein. The recombinant polypeptide, which was recognized by a monoclonal antibody directed against the native lectin, was purified from inclusion bodies after solubilization and refolding, using a combination of metal chelate and lactose affinity chromatography. The apparent molecular mass of the refolded polypeptide as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoreses was 16 kDa, whereas gel filtration chromatography clearly demonstrated that the recombinant protein, like its native counterpart, exists as a homodimer of two non-covalently associated subunits. Inhibition of haemagglutination suggests that the combining site of the recombinant lectin recognizes N-acetyl-galactosamine as the dominant sugar, thus confirming the correct folding of the monosaccharide combining site in the renatured lectin. To the best of our knowledge, this work represents the first reported detailed characterization of a recombinant lectin from apicomplexan parasites, and may contribute to a better understanding of the process of host cell recognition and invasion by these obligate intracellular protozoa.     

Crystallization of biologically active hemagglutinin-neuraminidase glycoprotein dimers proteolytically cleaved from human parainfluenza virus type 1.

We isolated, purified, and characterized the hemagglutinin-neuraminidase (HN) of human parainfluenza virus type 1, with the ultimate goal of producing crystals suitable for three-dimensional X-ray structure analysis. Pronase was used to cleave the globular head of the HN molecule directly from virus particles, forming HN monomers and dimers. The purified dimers retained neuraminidase and hemadsorption activity and were recognized by 14 anti-HN monoclonal antibodies, demonstrating intact HN antigenic structure and function. N-terminal sequence analysis of the dimers showed that cleavage had occurred at amino acid 136 or 137, freeing the C-terminal 438 or 439 amino acids. On electron micrography, the dimer appeared as two box-shaped structures, each approximately 5 by 5 nm. When the purified HN dimers were crystallized in hanging drops by vapor diffusion against 20% polyethylene glycol 3350, they formed both rectangular plates and needlelike crystals. The rectangular crystals diffracted X-rays, indicating an ordered atomic structure. However, the resolution was approximately 10 A (1 nm), insufficient for three-dimensional structural analysis. Experiments to improve the resolution by increasing the size and quality of the crystals are in progress.     

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.     

Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase

Paramyxoviruses are the main cause of respiratory disease in children. One of two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN), has several functions in addition to being the major surface antigen that induces neutralizing antibodies. Here we present the crystal structures of Newcastle disease virus HN alone and in complex with either an inhibitor or with the beta-anomer of sialic acid. The inhibitor complex reveals a typical neuraminidase active site within a beta-propeller fold. Comparison of the structures of the two complexes reveal differences in the active site, suggesting that the catalytic site is activated by a conformational switch. This site may provide both sialic acid binding and hydrolysis functions since there is no evidence for a second sialic acid binding site in HN. Evidence for a single site with dual functions is examined and supported by mutagenesis studies. The structure provides the basis for the structure-based design of inhibitors for a range of paramyxovirus-induced diseases.     

Influence of decorin and biglycan on mechanical properties of multiple tendons in knockout mice

Evaluations of tendon mechanical behavior based on biochemical and structural arrangement have implications for designing tendon specific treatment modalities or replacement strategies. In addition to the well studied type I collagen, other important constituents of tendon are the small proteoglycans (PGs). PGs have been shown to vary in concentration within differently loaded areas of tendon, implicating them in specific tendon function. This study measured the mechanical properties of multiple tendon tissues from normal mice and from mice with knock-outs of the PGs decorin or biglycan. Tail tendon fascicles, patellar tendons (PT), and flexor digitorum longus tendons (FDL), three tissues representing different in vivo loading environments, were characterized from the three groups of mice. It was hypothesized that the absence of decorin or biglycan would have individual effects on each type of tendon tissue. Surprisingly, no change in mechanical properties was observed for the tail tendon fascicles due to the PG knockouts. The loss of decorin affected the PT causing an increase in modulus and stress relaxation, but had little effect on the FDL. Conversely, the loss of biglycan did not significantly affect the PT, but caused a reduction in both the maximum stress and modulus of the FDL. These results give mechanical support to previous biochemical data that tendons likely are uniquely tailored to their specific location and function. Variances such as those presented here need to be further characterized and taken into account when designing therapies or replacements for any one particular tendon.