Viruses and viral proteins

For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes.

A carboxy terminal domain of the L protein of rinderpest virus possesses RNA triphosphatase activity - The first enzyme in the viral mRNA capping pathway

The large protein L of negative-sense RNA viruses is a multifunctional protein involved in transcription and replication of genomic RNA. It also possesses enzymatic activities involved in capping and methylation of viral mRNAs. The pathway for mRNA capping followed by the L protein of the viruses in the Morbillivirus genus has not been established, although it has been speculated that these viruses may follow the unconventional capping pathway as has been shown for some viruses of Rhabdoviridae family. We had earlier shown that the large protein L of Rinderpest virus expressed as recombinant L-P complex in insect cells as well as the ribonucleoprotein complex from purified virus possesses RNA triphosphatase (RTPase) and guanylyltransferase activities, in addition to RNA dependent RNA polymerase activity. In the present work, we demonstrate that RTPase as well as nucleoside triphosphatase (NTPase) activities are exhibited by a subdomain of the L protein in the C terminal region (a.a. 1640 1840). The RTPase activity depends absolutely on a divalent cation, either magnesium or manganese. Both the RTPase and NTPase activities of the protein show dual metal specificity. Two mutant proteins having alanine mutations in the glutamic acid residues in motif-A of the RTPase domain did not show RTPase activity, while exhibiting reduced NTPase activity suggesting overlapping active sites for the two enzymatic functions. The RTPase and NTPase activities of the L subdomain resemble those of the Vaccinia capping enzyme D1 and the baculovirus LEF4 proteins. (C) 2015 Elsevier Inc. All rights reserved.

Reconstruction of the Disassembly Pathway of an Icosahedral Viral Capsid and Shape Determination of Two Successive Intermediates

Viral capsids derived from an icosahedral plant virus widely used in physical and nanotechnological investigations were fully dissociated into dimers by a rapid change of pH. The process was probed in vitro at high spatiotemporal resolution by time-resolved small-angle X-ray scattering using a high brilliance synchrotron source. A powerful custom-made global fitting algorithm allowed us to reconstruct the most likely pathway parametrized by a set of stoichiometric coefficients and to determine the shape of two successive intermediates by ab initio calculations. None of these two unexpected intermediates was previously identified in self-assembly experiments, which suggests that the disassembly pathway is not a mirror image of the assembly pathway. These findings shed new light on the mechanisms and the reversibility of the assembly/disassembly of natural and synthetic virus-based systems. They also demonstrate that both the structure and dynamics of an increasing number of intermediate species become accessible to experiments.

New Keynesian Model Features that Can Reproduce Lead, Lag and Persistence Patterns

This paper uses a new method for describing dynamic comovement and persistence in economic time series which builds on the contemporaneous forecast error method developed in den Haan (2000). This data description method is then used to address issues in New Keynesian model performance in two ways. First, well known data patterns, such as output and inflation leads and lags and inflation persistence, are decomposed into forecast horizon components to give a more complete description of the data patterns. These results show that the well known lead and lag patterns between output and inflation arise mostly in the medium term forecasts horizons. Second, the data summary method is used to investigate a rich New Keynesian model with many modeling features to see which of these features can reproduce lead, lag and persistence patterns seen in the data. Many studies have suggested that a backward looking component in the Phillips curve is needed to match the data, but our simulations show this is not necessary. We show that a simple general equilibrium model with persistent IS curve shocks and persistent supply shocks can reproduce the lead, lag and persistence patterns seen in the data.

Persistence and Non-target Impact of Imazapyr Associated with Smooth Cordgrass Control in an Estuary

The herbicide (±-2-[4,5-dihydro-4-methyl-4-(1-methylethyl)- 5-oxo-1 H -imidazol-2-yl]-3-pyridinecarboxylic acid (imazapyr) has shown potential to control smooth cordgrass (Spartina alterniflora Loisel), a noxious weed in many estuaries throughout the world. Research was conducted under tidal estuary conditions in Willapa Bay, Washington, to determine imazapyr’s persistence and aquatic risk and impact to non-target estuary species. Persistence of imazapyr in water and sediment followed an exponential decay.(PDF has 6 pages.)

Longevity and Persistence of Triploid Grass Carp Stocked into the Santee Cooper Reservoirs of South Carolina

This study evaluated longevity and population persistence of 768,500 triploid grass carp (Ctenopharyngodon idella Valenciennes) stocked in the 70,000-ha Santee Cooper system in South Carolina from 1989 through 1996 to control hydrilla (Hydrilla verticillata (L.f.) Royle).

Long-Term Growth and Persistence with Endogenous Depreciation: Theory and Evidence

Previous research has shown a strong positive correlation between short-term persistence and long-term output growth as well as between depreciation rates and long-term output growth. This evidence, therefore, contradicts the standard predictions from traditional neoclassical or AK-type growth models with exogenous depreciation. In this paper, we first confirm these findings for a larger sample of 101 countries. We then study the dynamics of growth and persistence in a model where both the depreciation rate and growth are endogenous and procyclical. We find that the model s predictions become consistent with the empirical evidence on persistence, long-term growth and depreciation rates.

Design,optimization and in vivo evaluation of non-viral vectors for gene therapy. Applications in ocular disorders

227 págs.

Prevalence, intensity, longevity, and persistence of Anisakis sp. larvae and Lacistorhynchus tenuis metacestodes in San Francisco striped bass

Thirteen hundred and seventy-three striped bass, Marone saxatilis, were collected from the San Francisco Bay-Delta area to correlate host diet with parasitic infections and to determine the prevalence, intensity, longevity, and persistence of larval Anisakis sp. nematodes and the metacestode Lacistorhynchus tenuis. There is an increase in the prevalence and intensity of Anisakis sp. and in the intensity of L. tenuis with increase of age of the host. These increases are probably related to the diet and the persistence of tbe parasites. The infections of both species are overdispersed. San Francisco Bay striped bass are an incompatible host for both species of parasites. Degenerated Anisakis sp. will remain in lhe host for at least 8 months and L. tenuis metacestodes for 22 months. The occurrence of several other species of parasites and a tumor are also reported. (PDF file contains 10 pages.)

Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.

Radiobiological and biochemical investigations of polyoma virus-cell interactions

The cytolytic interaction of Polyoma virus with mouse embryo cells has been studied by radiobiological methods known to distinguish temperate from virulent bacteriophage. No evidence for "temperate" properties of Polyoma was found. During the course of these studies, it was observed that the curve of inactivation of Polyoma virus by ultraviolet light had two components - a more sensitive one at low doses, and a less sensitive one at higher doses. Virus which survives a low dose has an eclipse period similar to that of unirradiated virus, while virus surviving higher doses shows a significantly longer eclipse period. If Puromycin is present during the early part of the eclipse period, the survival curve becomes a single exponential with the sensitivity of the less sensitive component. These results suggest a repair mechanism in mouse cells which operates more effectively if virus development is delayed.

A comparison of the rates of inactivation of the cytolytic and transforming abilities of Polyoma by ultraviolet light, X-rays, nitrous acid treatment, or the decay of incorporated P³², showed that the transforming ability has a target size roughly 60% of that of the plaque-forming ability. It is thus concluded that only a fraction of the viral genes are necessary for causing transformation.

The appearance of virus-specific RNA in productively infected mouse kidney cells has been followed by means of hybridization between pulse-labelled RNA from the infected cells and the purified virus DNA. The results show a sharp increase in the amount of virus-specific RNA around the time of virus DNA synthesis. The presence of a small amount of virus-specific RNA in virus-free transformed cells has also been shown. This result offers strong evidence for the persistence of at least part of the viral genome in transformed cells.

Persistence of viable but non-culturable bacteria during the production and distribution of drinking water

The direct measurement of in situ respiring bacteria using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) shows that, especially for Gram-negative bacteria, large numbers of viable but non-culturable (VBNC) bacteria are present in finished water from a conventional water treatment plant, and the regrowth of bacteria along distribution networks can be seen rapidly by using this very sensitive technique. The level of bacterial inactivation with chlorine is much less important than has been previously supposed (based on experiments with non-injured laboratory strains of bacteria and classical culture techniques). Threshold values of VBNC bacteria leaving water treatment plants or regrowing along distribution systems have to be determined for better control of coliform regrowth and health- risks associated with the consumption of drinking water.

La aplicación empresarial del marketing viral y el efecto boca-oreja electrónico. Opiniones de las empresas

[ES] La práctica tradicional del boca-oreja, natural desde hace años como modo de publicidad efectiva sin recurrir a grandes inversiones ni contrataciones en medios masivos se ha convertido en una nueva posibilidad de marketing con la llegada de las nuevas tecnologías e Internet. Así ha aparecido una nueva estrategia de marketing que consiste en explotar las redes sociales preexistentes para producir incrementos exponenciales en conocimiento de marca, denominada marketing viral. El efecto "boca-oreja online" que genera es una herramienta poderosa para las empresas, aunque su verdadero potencial está aún por descubrir. El objetivo del presente trabajo de investigación es analizar este fenómeno a través de una amplia revisión bibliográfica del término, así como un estudio empírico consistente en una entrevista en profundidad realizada a una muestra de importantes empresas españolas. Los resultados indican que el marketing viral puede ser empleado para el beneficio, tanto de grandes empresas con grandes presupuestos, como de pequeños negocios. Las experiencias exitosas de su utilización demuestran que, cuando se usa integrado en el resto de estrategias comerciales de la empresa, puede mejorar la recomendación de la marca e incrementar su notoriedad en el mercado. El marketing viral puede ser ventajoso a la hora de lanzar un nuevo producto al mercado, sin embargo, la efectividad y la medición de las campañas son vistas por muchos académicos y profesionales como un punto débil de la estrategia.

Assembly of viral genomes from metagenomes

Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity are, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.

HIV-1 Capture and Transmission by Dendritic Cells: The Role of Viral Glycolipids and the Cellular Receptor Siglec-1

Dendritic cells (DCs) are essential in order to combat invading viruses and trigger antiviral responses. Paradoxically, in the case of HIV-1, DCs might contribute to viral pathogenesis through trans-infection, a mechanism that promotes viral capture and transmission to target cells, especially after DC maturation. In this review, we highlight recent evidence identifying sialyllactose-containing gangliosides in the viral membrane and the cellular lectin Siglec-1 as critical determinants for HIV-1 capture and storage by mature DCs and for DC-mediated trans-infection of T cells. In contrast, DC-SIGN, long considered to be the main receptor for DC capture of HIV-1, plays a minor role in mature DC-mediated HIV-1 capture and trans-infection.