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Background: Avian influenza virus (AIV) is classified into two pathotypes, low pathogenic (LP) and high pathogenic ( HP), based on virulence in chickens. Differences in pathogenicity between HPAIV and LPAIV might eventually be related to specific characteristics of strains, tissue tropism and host responses. Methods: To study differences in disease development between HPAIV and LPAIV, we examined the first appearance and eventual load of viral RNA in multiple organs as well as host responses in brain and intestine of chickens infected with two closely related H7N1 HPAIV or LPAIV strains. Results: Both H7N1 HPAIV and LPAIV spread systemically in chickens after a combined intranasal/intratracheal inoculation. In brain, large differences in viral RNA load and host gene expression were found between H7N1 HPAIV and LPAIV infected chickens. Chicken embryo brain cell culture studies revealed that both HPAIV and LPAIV could infect cultivated embryonic brain cells, but in accordance with the absence of the necessary proteases, replication of LPAIV was limited. Furthermore, TUNEL assay indicated apoptosis in brain of HPAIV infected chickens only. In intestine, where endoproteases that cleave HA of LPAIV are available, we found minimal differences in the amount of viral RNA and a large overlap in the transcriptional responses between HPAIV and LPAIV infected chickens. Interestingly, brain and ileum differed clearly in the cellular pathways that were regulated upon an AI infection. Conclusions: Although both H7N1 HPAIV and LPAIV RNA was detected in a broad range of tissues beyond the respiratory and gastrointestinal tract, our observations indicate that differences in pathogenicity and mortality between HPAIV and LPAIV could originate from differences in virus replication and the resulting host responses in vital organs like the brain.

This contribution describes the metal ion responsive adhesion of vesicles induced by a conformational switch of a non-covalent linker molecule. A p-tert-butylbenzyl dimer with a flexible N,N'-bis(3-aminopropyl)ethylenediamine spacer was used as a non-covalent linker, which induces aggregation and adhesion (but not fusion) of host bilayer vesicles composed of amphiphilic beta-cyclodextrins by the formation of hydrophobic inclusion complexes. The aggregation and adhesion of the vesicles in dilute aqueous solution was confirmed by isothermal titration calorimetry (ITC), optical density measurements at 600 nm (OD600), dynamic light scattering (DLS), zeta-potential measurements, cryogenic transmission electron microscopy (cryo-TEM) and fluorescence spectroscopy. However, in the presence of a divalent metal ion like Cu(2+), the tetra-amine linker molecule forms a stable metal coordination complex and dramatically switches its conformation from linear to bent, which results in the dissociation of intervesicular complexes, and leads to the dispersion of vesicle clusters. This process is reversible in the presence of a strong metal ion chelator, such as EDTA, that scavenges the Cu(2+) ion complexed by the linker. The linker molecule regains its linear conformation and triggers the reaggregation of the vesicles. In contrast, conformational switching was inhibited by introducing a rigid N,N'-bis(3-aminopropyl)piperazine spacer in the non-covalent linker molecule and vesicles do not aggregate in the presence of a cyclic guest that can only bind intravesicularly. Thus, a metal ion regulated molecular switch can control the aggregation state of an organic colloidal solution.

Two distinct envelope fusion proteins (EFPs) (GP64 and F) have been identified in members of the Baculoviridae family of viruses. F proteins are found in group II nucleopolyhedroviruses (NPVs) of alphabaculoviruses and in beta- and deltabaculoviruses, while GP64 occurs only in group I NPVs of alphabaculoviruses. It was proposed that an ancestral baculovirus acquired the gp64 gene that conferred a selective advantage and allowed it to evolve into group I NPVs. The F protein is a functional analogue of GP64, as evidenced from the rescue of gp64-null Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV) by F proteins from group II NPVs or from betabaculoviruses. However, GP64 failed to rescue an F-null Spodoptera exigua MNPV (SeMNPV) (group II NPV). Here, we report the successful generation of an infectious gp64-rescued group II NPV of Helicoverpa armigera (vHaBac Delta F-gp64). Viral growth curve assays and quantitative real-time PCR (Q-PCR), however, showed substantially decreased infectivity of vHaBac Delta F-gp64 compared to the HaF rescue control virus vHaBac Delta F-HaF. Electron microscopy further showed that most vHaBac Delta F-gp64 budded viruses (BV) in the cell culture supernatant lacked envelope components and contained morphologically aberrant nucleocapsids, suggesting the improper BV envelopment or budding of vHaBac Delta F-gp64. Bioassays using pseudotyped viruses with a reintroduced polyhedrin gene showed that GP64-pseudotyped Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) significantly delayed the mortality of infected H. armigera larvae.

We report a bicontinuous cubic phase forming in the presence of the Influenza HA fusion peptide in coarse grained molecular dynamics simulations. Starting from a random mixture of DOPE, water, and fusion peptides, we observe spontaneous formation of a stable bicontinuous phase. Unlike all previously reported bicontinuous cubic phases the one formed in our simulations is a single phase in the sense that there are no multiple isolated compartments of water or lipid.

F proteins of group II nucleopolyhedroviruses (NPVs) are envelope fusion proteins essential for virus entry and egress. An F-null Helicoverpa armigera single nucleocapsid NPV (HearNPV) bacmid, HaBacF, was constructed. This bacmid could not produce infectious budded virus (BV) when transfected into HzAM1 cells, showing that F protein is essential for cell-to-cell transmission of BVs. When HaBacF was pseudotyped with the homologous F protein (HaBacF-HaF, positive control) or with the heterologous F protein from Spodoptera exigua multinucleocapsid NPV (SeMNPV) (HaBacF-SeF), infectious BVs were produced with similar kinetics. In the late phase of infection, the BV titre of HaBacF-SeF virus was about ten times lower than that of HaBacF-HaF virus. Both pseudotyped viruses were able to fuse HzAM1 cells in a similar fashion. The F proteins of both HearNPV and SeMNPV were completely cleaved into F1 and F2 in the BVs of vHaBacF-HaF and vHaBacF-SeF, respectively, but the cleavage of SeF in vHaBacF-SeF-infected HzAM1 cells was incomplete, explaining the lower BV titre of vHaBacF-SeF. Polyclonal antisera against HaF1 and SeF1 specifically neutralized the infection of vHaBacF-HaF and vHaBacF-SeF, respectively. HaF1 antiserum showed some cross-neutralization with vHaBacF-SeF. These results demonstrate that group II NPV F proteins can be functionally replaced with a homologue of other group II NPVs, suggesting that the interaction of F with other viral or host proteins is not absolutely species-specific.

Group II nucleopolyhedroviruses (NPVs), e.g. Helicoverpa armigera (Hear) NPV and Spodoptera exigua (Se) MNPV (multiple NPV), lack a GP64-like protein that is present in group I NPVs, e.g. Autographa californica (Ac)MNPV, but have an unrelated envelope fusion protein named F. Three AcMNPV viruses were constructed by introducing AcMNPV gp64, HearNPV f or SeMNPV f genes, respectively, into a gp64-negative AcMNPV bacmid. Sf21 cells were incubated with different amounts of inactivated budded virus to occupy receptors and were subsequently infected with a fixed amount of infectious virus to compete for attachment. The results suggest that GP64 and F act on their own and use different receptors, while the two different F proteins exploit the same receptor. Additionally, gp64-null AcMNPV pseudotyped with baculovirus F was, in contrast to GP64, unable to transduce mammalian cells, indicating that mammalian cells do not possess baculovirus F protein receptors despite the structural similarity of baculovirus F to vertebrate viral fusion proteins.

The F protein is the major glycoprotein present in the envelopes of budded virus (BV) of members of the family Baculoviridae. The F protein mediates low-pH-activated fusion with insect cell membranes. Baculovirus F proteins are synthesized as a precursor (F0) and cleaved post-translationally into two disulfide-bonded subunits, F1 (C-terminal, large subunit) and F2 (N-terminal, small subunit). Recently, N-linked glycosylation of the F1 and F2 subunits of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was demonstrated [Long, G., Westenberg, M., Wang, H., Vlak, J. M. & Hu, Z. (2006). J Gen Virol 87, 839¿846]. Sequence analysis frequently predicts that one or more N-linked glycosylation sites are present in the F2 subunit of baculovirus F proteins. N-glycans on envelope fusion proteins are usually required for proper conformational integrity and biological function, such as infectivity. This study examined the importance of N-linked glycosylation of the F2 subunit of HearNPV by site-directed mutagenesis. The only putative N-linked glycosylation site in F2 was eliminated by mutating asparagine (N104) to glutamine (Q), resulting in the mutant HearNPVfN104Q. When inserted into an f-null HearNPV and a gp64-null bacmid of Autographa californica multiple nucleopolyhedrovirus, infectious BV could be retrieved that contained unglycosylated F2. The virulence of HearNPVfN104Q was enhanced, as BV was produced earlier after infection and yielded larger plaques than f-null HearNPV repaired with the wild-type f gene. HearNPVfN104Q BV also induced much more efficient low-pH-activated syncytium formation. These results indicate that N-linked glycosylation of the HearNPV baculovirus F2 subunit is not essential for viral infectivity and suggest that it is involved in BV production and fusogenicity

F proteins from baculovirus nucleopolyhedrovirus (NPV) group II members are the major budded virus (BV) viral envelope fusion proteins. They undergo furin-like proteolysis processing in order to be functional. F proteins from different baculovirus species have a long cytoplasmic tail domain (CTD), ranging from 48 (Spodoptera litura multicapsid NPV [MNPV]) to 78 (Adoxophyes honmai NPV) amino acid (aa) residues, with a nonassigned function. This CTD is much longer than the CTD of GP64-like envelope fusion proteins (7 aa), which appear to be nonessential for BV infectivity. Here we have investigated the functional role of the CTD of Helicoverpa armigera single-capsid NPV (HearNPV), a group II NPV. We combined a newly constructed HearNPV f-null bacmid knockout-repair system and an Autographa californica MNPV (AcMNPV) gp64-null bacmid knockout-pseudotype system with mutation and rescue experiments to study the functional role of the baculovirus F protein CTD. We show that except for the 16 C-terminal aa, the HearNPV F CTD is essential for virus spread from cell to cell. In addition, the CTD of HearNPV F is involved in BV production in a length-dependent manner and is essential for BV infectivity. The tyrosine residue Y658, located 16 aa from the C terminus, seems to be critical. However, HearNPV F without a CTD still rescues the infectivity of gp64-null AcMNPV BV, indicating that the CTD is not involved in processing and fusogenicity. Altogether, our results indicate that the F protein is essential for baculovirus BV infectivity and that the CTD is important for F protein incorporation into BV.

Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2a, E, GP3, and GP4. Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indicating that these proteins interact with each other and are assembled into virions as a multimeric complex. Independent evidence for such complexes was obtained by coexpression of the minor envelope proteins in BHK-21 cells using a Semliki Forest virus expression system. By analyzing the maturation of their N-linked oligosaccharides, we found that the glycoproteins were each retained in the endoplasmic reticulum unless expressed together, in which case they were collectively transported through the Golgi complex to the plasma membrane and were even detected in the extracellular medium. As the PRRSV particles lacking the minor envelope proteins are not infectious, we hypothesize that the virion surface structures formed by these proteins function in viral entry by mediating receptor binding and/or virus-cell fusion

Group II nucleopolyhedroviruses (NPVs), e.g., Spodoptera exigua MNPV, lack a GP64-like protein that is present in group I NPVs but have an unrelated envelope fusion protein named F. In contrast to GP64, the F protein has to be activated by a posttranslational cleavage mechanism to become fusogenic. In several vertebrate viral fusion proteins, the cleavage activation generates a new N terminus which forms the so-called fusion peptide. This fusion peptide inserts in the cellular membrane, thereby facilitating apposition of the viral and cellular membrane upon sequential conformational changes of the fusion protein. A similar peptide has been identified in NPV F proteins at the N terminus of the large membrane-anchored subunit F-1. The role of individual amino acids in this putative fusion peptide on viral infectivity and propagation was studied by mutagenesis. Mutant F proteins with single amino acid changes as well as an F protein with a deleted putative fusion peptide were introduced in gp64-null Autographa californica MNPV budded viruses (BVs). None of the mutations analyzed had an major effect on the processing and incorporation of F proteins in the envelope of BVs. Only two mutants, one with a substitution for a hydrophobic residue (F152R) and one with a deleted putative fusion peptide, were completely unable to rescue the gp64-null mutant. Several nonconservative substitutions for other hydrophobic residues and the conserved lysine residue had only an effect on viral infectivity. In contrast to what was expected from vertebrate virus fusion peptides, alanine substitutions for glycines did not show any effect.

Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of syntaxin 1, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state., QC 20100525

Cryo-transmission electron microscopy has been applied to the study of the changes induced by phospholipase C on large unilamellar vesicles containing phosphatidylcholine, as well as to the action of sphingomyelinase on vesicles containing sphingomyelin. In both cases vesicle aggregation occurs as the earliest detectable phenomenon; later, each system behaves differently. Phospholipase C induces vesicle fusion through an intermediate consisting of aggregated and closely packed vesicles (the ''honeycomb structure'') that finally transforms into large spherical vesicles. The same honeycomb structure is also observed in the absence of enzyme when diacylglycerols are mixed with the other lipids in organic solution, before hydration. In this case the sample then evolves toward a cubic phase. The fact that the same honeycomb intermediate can lead to vesicle fusion (with enzyme-generated diacylglycerol) or to a cubic phase (when diacylglycerol is premixed with the lipids) is taken in support of the hypothesis according to which a highly curved lipid structure (''stalk'') would act as a structural intermediate in membrane fusion, Sphingomyelinase produces complete leakage of vesicle aqueous contents and an increase in size of about one-third of the vesicles. A mechanism of vesicle opening and reassembling is proposed in this case., Addresses: UNIV PAIS VASCO, DEPT BIOQUIM, GRP BIOMEMBRANES, CSIC, E-48080 BILBAO, SPAIN. UNIV UPPSALA, DEPT PHYS CHEM, S-75121 UPPSALA, SWEDEN.

During the past few years cryotransmission electron microscopy (EM) of vitrified thin samples has gained acceptance as a standard method in the arsenal of the colloid and interface scientist. The seemingly direct visualization of fluid colloidal structures during the use of cryotransmission EM is both convincing and reliable to the scientist who nowadays has an increasing awareness of the limitations and pitfalls of instrumentation. Notable recent observations include branched threadlike micelles, faceted particles (cubosomes) of a dispersed cubic phase and transitions of certain structures from globular micelles via bilayers to reversed structures. These transitions may be caused by changes of compos ition, temperature, pH, or salt concentration., Addresses: Almgren M, UNIV UPPSALA, DEPT PHYS CHEM, BOX 532, S-75121 UPPSALA, SWEDEN.