Rapid Neutrophil Response Controls Fast-Replicating Intracellular Bacteria but Not Slow-Replicating Mycobacterium tuberculosis

Being one of the first cells to invade the site of infection, neutrophils play an important role in the control of various bacterial and viral infections. In the present work, the contribution of neutrophils to the control of infection with different intracellular bacteria was investigated. Mice were treated with the neutrophil-depleting monoclonal antibody RB6-8C5, and the time course of infection in treated and untreated mice was compared by using intracellular bacterial species and strains varying in virulence and replication rate. The results indicate that neutrophils are crucial for the control of fast-replicating intracellular bacteria, whereas early neutrophil effector mechanisms are dispensable for the control of the slow-replicating Mycobacterium tuberculosi

Structural Studies of West Nile Virus in Complex with Neutralizing Antibodies.

West Nile virus (WNV) is a positive strand RNA virus in the family Flaviviridae, which includes members such as dengue, Japanese encephalitis, tick-borne encephalitis, yellow fever and Hepatitis C. As with other members of the genus, it is arthropod transmitted and has recently established itself as an endemic virus in the United States. Although most infections are asymptomatic, clinical manifestations of WNV include encephalitis and death. We have been interested in investigating the nature of the immune response with particular emphasis on the role of antibodies in reducing the level of infection. We have used a combination of techniques, but primarily structure, as a tool to probe the nature of antibody-mediated virus neutralization. Our results suggest that neutralization of virus particles is more complex than originally envisioned, with multiple mechanisms utilized. Using a combination of X-ray crystallography and cryo-electron microscopy, several virus-antibody complexes have been determined at pseudo-atomic resolution. These studies suggest the following: 1) flavivirus particles exhibit dynamic motions or breathing that transiently expose cryptic epitopes; 2) although there are 180 potential binding sites for each monoclonal antibody the quasi-equivalent nature of the virion usually permits only a subset of sites to be utilized; 3) the availability of these sites, the epitope itself, and the avidity of antibody directly influence the mechanism of neutralization; and 4) particles thought to be incapable of infecting cells, so-called immature viruses, may play a critical role in immune surveillance and reactivity. The structure of the flavivirus virion and complexes of monoclonal antibodies will be presented along with data to support mechanisms antibody-mediated flavivirus neutralizatio

Capturing a Flavivirus Pre-Fusion Intermediate

During cell entry of flaviviruses, low endosomal pH triggers the rearrangement of the viral surface glycoproteins to a fusion-active state that allows the release of the infectious RNA into the cytoplasm. In this work, West Nile virus was complexed with Fab fragments of the neutralizing mAb E16 and was subsequently exposed to low pH, trapping the virions in a pre-fusion intermediate state. The structure of the complex was studied by cryo-electron microscopy and provides the first structural glimpse of a flavivirus fusion intermediate near physiological conditions. A radial expansion of the outer protein layer of the virion was observed compared to the structure at pH 8. The resulting ∼60 Å-wide shell of low density between lipid bilayer and outer protein layer is likely traversed by the stem region of the E glycoprotein. By using antibody fragments, we have captured a structural intermediate of a virus that likely occurs during cell entry. The trapping of structural transition states by antibody fragments will be applicable for other processes in the flavivirus life cycle and delineating other cellular events that involve conformational rearrangements

A Novel Substrate-Based HIV-1 Protease Inhibitor Drug Resistance Mechanism

BACKGROUND: HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors. METHODS AND FINDINGS: We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy. CONCLUSIONS: HIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy

In vitro-Untersuchungen zu Parvovirus B19-Proteinkapsiden und deren Rezeptor-Interaktionen

Parvovirus B19 ist der einzige bekannte humanpathogene Vertreter der Parvoviridae. Ziel der vorliegenden Arbeit war die detaillierte biophysikalische Beschreibung der Interaktion von B19 mit seinem vorgeschlagenen, zellulären Rezeptor, dem Glycospingolipid Globosid. Es wurde eine spezifische IgG-Fraktion gegen das virale Strukturprotein VP2 zum Einsatz als immobilisierender Träger für Bindungsassays hergestellt und die Präparation rekombinanter VP2-Proteinkapside optimiert. Zur Charakterisierung der B19/Globosid-Wechselwirkung wurden Bindungstests mit fluoreszenz-markierten Liposomen und 125I-markierten Kapsiden etabliert. Die direkten Testsysteme lieferten keine Hinweise auf eine spezifische Kapsid/Globosid-Interaktion. Die Ergebnisse konnten durch Biosensormessungen und kalorimetrische Untersuchungen verifiziert werden. Hämagglutinationsuntersuchungen im Rahmen dieser Arbeit bestätigten die publizierte Hemmaktivität von Globosid, zeigten aber eine vergleichbare Aktivität von Globopentaosylceramid. Dies stellt eine hohe Spezifität des Globosid-Kapsid-Kontaktes in Frage. Die Ergebnisse implizieren, daß Globosid nicht als singulärer Rezeptor für B19 agieren kann, aber vielleicht im Kontext einer Interaktion des Virus mit einem anderen Rezeptormolekül erkannt wird. Daher wurden erste Untersuchungen zur Identifizierung anderer Rezeptormoleküle in Membranproteinfraktionen von humanen Zellinien durchgeführt und die Bindung 125I-markierter Kapside an mehrere Proteine von 30 bis 100 kD Größe detektiert. Die Identität der Banden ist weitgehend unklar, wobei die Spezifität einer Interaktion mit Carboanhydrase noch überprüft werden muß. Das Kapsid von Parvovirus B19 besteht aus nur zwei Strukturproteinen. Für weitere Bindungstests und Strukturuntersuchungen wurden heterogene VP1/VP2-Partikel im Baculo/Sf9-System produziert. Durch Mutation des Promoterbereiches des bicistronischen Baculovirus konnten Mischkapside mit VP1-Anteilen von 45 bzw. 30 hergestellt werden

The VP1 Unique Region of Parvovirus B19 and Its Constituent Phospholipase A2-Like Activity

Parvovirus B19 is the causative agent of erythema infectiosum. In addition, parvovirus B19 infection may be associated with other disease manifestations, namely, thrombocytopenia or granulocytopenia, spontaneous abortion or hydrops fetalis in pregnant women, acute and chronic arthritis, and systemic lupus erythematosus. Based on sequence homology data, a phospholipase A2 motif has been identified in the VP1 unique region of parvovirus B19. (Y. Li et al., J. Gen. Virol. 82:2821-2825, 2001; Z. Zadori et al., Dev. Cell 1:291-302, 2001). We have established a new in vitro assay based on electrospray ionization tandem mass spectroscopy to show that phospholipase A2 activity is present in the VP1 unique region produced in Escherichia coli and in virus-like particles consisting of combinations of VP1 and VP2 proteins expressed by recombinant baculovirus. The enzyme activity of the VP1 unique region showed typical Ca(2+) dependency and could be inhibited by manoalide and 4-bromophenacylbromide, which bind covalently to lysine and histidine residues, respectively, as part of the active center of the enzyme. By using subfragments, we demonstrated an association between the phospholipase A2-like activity and the carboxy-terminal domain of the VP1 unique region