bluuebunny/crossref_metadata_2025 huggingface dataset:
An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region
Authors: Schweizer Nina , Pawar Nisha , Weiss Matthias , Maiato Helder
Published: August 2015

The mitotic spindle is a microtubular assembly required for chromosome segregation during mitosis. Additionally, a spindle matrix has long been proposed to assist this process, but its nature has remained elusive. By combining live-cell imaging with laser microsurgery, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy in Drosophila melanogaster S2 cells, we uncovered a microtubule-independent mechanism that underlies the accumulation of molecules in the spindle region. This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells. Supported by mathematical modeling, we demonstrate that organelle exclusion by a membrane system causes spatio-temporal differences in molecular crowding states that are sufficient to drive accumulation of mitotic regulators, such as Mad2 and Megator/Tpr, as well as soluble tubulin, in the spindle region. This membranous “spindle envelope” confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation. Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function.
Late steps in cytoplasmic maturation of assembly-competent axonemal outer arm dynein in Chlamydomonas require interaction of ODA5 and ODA10 in a complex
Authors: Dean Anudariya B. , Mitchell David R.
Published: August 2015

Axonemal dyneins are multisubunit enzymes that must be preassembled in the cytoplasm, transported into cilia by intraflagellar transport, and bound to specific sites on doublet microtubules, where their activity facilitates microtubule sliding-based motility. Outer dynein arms (ODAs) require assembly factors to assist their preassembly, transport, and attachment to cargo (specific doublet A-tubule sites). In Chlamydomonas, three assembly factors—ODA5, ODA8, and ODA10—show genetic interactions and have been proposed to interact in a complex, but we recently showed that flagellar ODA8 does not copurify with ODA5 or ODA10. Here we show that ODA5 and ODA10 depend on each other for stability and coexist in a complex in both cytoplasmic and flagellar extracts. Immunofluorescence and immuno–electron microscopy reveal that ODA10 in flagella localizes strictly to a proximal region of doublet number 1, which completely lacks ODAs in Chlamydomonas. Studies of the in vitro binding of ODAs to axonemal doublets reveal a role for the ODA5/ODA10 assembly complex in cytoplasmic maturation of ODAs into a form that can bind to doublet microtubules.
Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses
Authors: Lehmann Kathleen C. , Gulyaeva Anastasia , Zevenhoven-Dobbe Jessika C. , Janssen George M. C. , Ruben Mark , Overkleeft Hermen S. , van Veelen Peter A. , Samborskiy Dmitry V. , Kravchenko Alexander A. , Leontovich Andrey M. , Sidorov Igor A. , Snijder Eric J. , Posthuma Clara C. , Gorbalenya Alexander E.
Published: August 2015

RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is N-terminally adjacent to the RdRp and has no apparent homologs elsewhere. Based on its conservation profile, this domain is proposed to have nucleotidylation activity. We used recombinant non-structural protein 9 of the arterivirus equine arteritis virus (EAV) and different biochemical assays, including irreversible labeling with a GTP analog followed by a proteomics analysis, to demonstrate the manganese-dependent covalent binding of guanosine and uridine phosphates to a lysine/histidine residue. Most likely this was the invariant lysine of the newly identified domain, named nidovirus RdRp-associated nucleotidyltransferase (NiRAN), whose substitution with alanine severely diminished the described binding. Furthermore, this mutation crippled EAV and prevented the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in cell culture, indicating that NiRAN is essential for nidoviruses. Potential functions supported by NiRAN may include nucleic acid ligation, mRNA capping and protein-primed RNA synthesis, possibilities that remain to be explored in future studies.
Bubbles with shock waves and ultrasound: a review
Authors: Ohl Siew-Wan , Klaseboer Evert , Khoo Boo Cheong
Published: August 2015

The study of the interaction of bubbles with shock waves and ultrasound is sometimes termed ‘acoustic cavitation'. It is of importance in many biomedical applications where sound waves are applied. The use of shock waves and ultrasound in medical treatments is appealing because of their non-invasiveness. In this review, we present a variety of acoustics–bubble interactions, with a focus on shock wave–bubble interaction and bubble cloud phenomena. The dynamics of a single spherically oscillating bubble is rather well understood. However, when there is a nearby surface, the bubble often collapses non-spherically with a high-speed jet. The direction of the jet depends on the ‘resistance' of the boundary: the bubble jets towards a rigid boundary, splits up near an elastic boundary, and jets away from a free surface. The presence of a shock wave complicates the bubble dynamics further. We shall discuss both experimental studies using high-speed photography and numerical simulations involving shock wave–bubble interaction. In biomedical applications, instead of a single bubble, often clouds of bubbles appear (consisting of many individual bubbles). The dynamics of such a bubble cloud is even more complex. We shall show some of the phenomena observed in a high-intensity focused ultrasound (HIFU) field. The nonlinear nature of the sound field and the complex inter-bubble interaction in a cloud present challenges to a comprehensive understanding of the physics of the bubble cloud in HIFU. We conclude the article with some comments on the challenges ahead.
Anatomy of a negative feedback loop: the case of I
κ
B
α
Authors: Fagerlund Riku , Behar Marcelo , Fortmann Karen T. , Lin Y. Eason , Vargas Jesse D. , Hoffmann Alexander
Published: August 2015

The magnitude, duration and oscillation of cellular signalling pathway responses are often limited by negative feedback loops, defined as an ‘activator-induced inhibitor’ regulatory motif. Within the NF
κ
B signalling pathway, a key negative feedback regulator is I
κ
B
α
. We show here that, contrary to current understanding, NF
κ
B-inducible expression is not sufficient for providing effective negative feedback. We then employ computational simulations of NF
κ
B signalling to identify I
κ
B
α
molecular properties that are critical for proper negative feedback control and test the resulting predictions in biochemical and single-cell live-imaging studies. We identified nuclear import and nuclear export of I
κ
B
α
and the I
κ
B
α
–NF
κ
B complex, as well as the free I
κ
B
α
half-life, as key determinants of post-induction repression of NF
κ
B and the potential for subsequent reactivation. Our work emphasizes that negative feedback is an emergent systems property determined by multiple molecular and biophysical properties in addition to the required ‘activator-induced inhibitor’ relationship.
Connectivity, passability and heterogeneity interact to determine fish population persistence in river networks
Authors: Samia Yasmine , Lutscher Frithjof , Hastings Alan
Published: August 2015

The movement of fish in watersheds is frequently inhibited by human-made migration barriers such as dams or culverts. The resulting lack of connectivity of spatial subpopulations is often cited as a cause for observed population decline. We formulate a matrix model for a spatially distributed fish population in a watershed, and we investigate how location and other characteristics of a single movement barrier impact the asymptotic growth rate of the population. We find that while population growth rate often decreases with the introduction of a movement obstacle, it may also increase due to a ‘retention effect’. Furthermore, obstacle mortality greatly affects population growth rate. In practice, different connectivity indices are used to predict population effects of migration barriers, but the relation of these indices to population growth rates in demographic models is often unclear. When comparing our results with the dentritic connectivity index, we see that the index captures neither the retention effect nor the influences of obstacle mortality. We argue that structural indices cannot entirely replace more detailed demographic models to understand questions of persistence and extinction. We advocate the development of novel functional indices and characteristics.
The role of passive avian head stabilization in flapping flight
Authors: Pete Ashley E. , Kress Daniel , Dimitrov Marina A. , Lentink David
Published: August 2015

Birds improve vision by stabilizing head position relative to their surroundings, while their body is forced up and down during flapping flight. Stabilization is facilitated by compensatory motion of the sophisticated avian head–neck system. While relative head motion has been studied in stationary and walking birds, little is known about how birds accomplish head stabilization during flapping flight. To unravel this, we approximate the avian neck with a linear mass–spring–damper system for vertical displacements, analogous to proven head stabilization models for walking humans. We corroborate the model's dimensionless natural frequency and damping ratios from high-speed video recordings of whooper swans (
Cygnus cygnus
) flying over a lake. The data show that flap-induced body oscillations can be passively attenuated through the neck. We find that the passive model robustly attenuates large body oscillations, even in response to head mass and gust perturbations. Our proof of principle shows that bird-inspired drones with flapping wings could record better images with a swan-inspired passive camera suspension.
Heparan sulfate and heparin interactions with proteins
Authors: Meneghetti Maria C. Z. , Hughes Ashley J. , Rudd Timothy R. , Nader Helena B. , Powell Andrew K. , Yates Edwin A. , Lima Marcelo A.
Published: August 2015

Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during
in vitro
,
ex vivo
and
in vivo
experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure–activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure–activity relationships and regulation will be discussed.
The brain uses extrasomatic information to estimate limb displacement
Authors: Terekhov Alexander V. , Hayward Vincent
Published: August 2015

A fundamental problem faced by the brain is to estimate whether a touched object is rigidly attached to a ground reference or is movable. A simple solution to this problem would be for the brain to test whether pushing on the object with a limb is accompanied by limb displacement. The mere act of pushing excites large populations of mechanoreceptors, generating a sensory response that is only weakly sensitive to limb displacement if the movements are small, and thus can hardly be used to determine the mobility of the object. In the mechanical world, displacement or deformation of objects frequently co-occurs with microscopic fluctuations associated with the frictional sliding of surfaces in contact or with micro-failures inside an object. In this study, we provide compelling evidence that the brain relies on these microscopic mechanical events to estimate the displacement of the limb in contact with an object, and hence the mobility of the touched object. We show that when pressing with a finger on a stiff surface, fluctuations that resemble the mechanical response of granular solids provoke a sensation of limb displacement. Our findings suggest that when acting on an external object, prior knowledge about the sensory consequences of interacting with the object contributes to proprioception.
Are there reliable constitutive laws for dynamic friction?
Authors: Woodhouse Jim , Putelat Thibaut , McKay Andrew
Published: August 2015

Structural vibration controlled by interfacial friction is widespread, ranging from friction dampers in gas turbines to the motion of violin strings. To predict, control or prevent such vibration, a constitutive description of frictional interactions is inevitably required. A variety of friction models are discussed to assess their scope and validity, in the light of constraints provided by different experimental observations. Three contrasting case studies are used to illustrate how predicted behaviour can be extremely sensitive to the choice of frictional constitutive model, and to explore possible experimental paths to discriminate between and calibrate dynamic friction models over the full parameter range needed for real applications.
Protection and antibody responses in different strains of mouse immunized with plasmid DNAs encoding influenza virus haemagglutinin, neuraminidase and nucleoprotein
Authors: Chen Ze , Yoshikawa Tomoki , Kadowaki Shin-etsu , Hagiwara Yukari , Matsuo Kazutoshi , Asanuma Hideki , Aizawa Chikara , Kurata Takeshi , Tamura Shin-ichi
Published: August 2015

Protection against influenza virus infection and antibody responses in mice vaccinated with plasmid DNAs encoding haemagglutinin (HA), neuraminidase (NA) and nucleoprotein (NP) were compared among BALB/c (H-2d), B10 (H-2b) and C3H (H-2k) mice. Mice were inoculated with each DNA construct twice, 3 weeks apart, at a dose of 1 μg per mouse by particle-mediated DNA transfer (gene gun) to the epidermis. They were challenged with a lethal dose of the homologous virus 7 days after the second vaccination. NA-DNA provided significant protection in all strains of mouse, whereas HA-DNA afforded significant protection only in BALB/c mice. The serum antibody titres against NA or HA molecules in BALB/c, C3H and B10 mice were high, intermediate and low, respectively. NP-DNA failed to provide protection in any strain of mouse, and elicited low titres of anti-NP antibodies. These results suggest that NA-DNA can be used as a vaccine component to provide effective protection against influenza virus infection in various strains of mouse.
Human herpesvirus-8-encoded LNA-1 accumulates in heterochromatin- associated nuclear bodies
Authors: Szekely Laszlo , Kiss Csaba , Mattsson Karin , Kashuba Elena , Pokrovskaja Katja , Juhasz Attila , Holmvall Pia , Klein George
Published: August 2015

Subnuclear distribution of the human herpesvirus-8 (HHV-8)- encoded nuclear protein LNA-1 was analysed at high resolution in body cavity (BC) lymphoma-derived cell lines, in cell hybrids between BC cells and various human and mouse cells and in freshly infected K562 and ECV cell lines. Three-dimensional reconstruction of nuclei from optical sections and quantitative analysis of the distribution of LNA-1 fluorescence in relation to chromatin showed that LNA-1 associates preferentially with the border of heterochromatin in the interphase nuclei. This was further confirmed in the following systems: in endo- and exonuclease-digested nuclei, in human–mouse (BC-1–Sp2- 0) hybrids and on chromatin spreads. LNA-1 was found to bind to mitotic chromosomes at random. Epstein–Barr virus (EBV), but not HHV-8, was rapidly lost from mouse–human hybrid cells in parallel with the loss of human chromosomes. HHV-8 could persist on the residual mouse background for more than 8 months. In early human–mouse hybrids that contain a single fused nucleus, LNA-1 preferentially associates with human chromatin. After the gradual loss of the human chromosomes, LNA-1 becomes associated with the murine pericentromeric heterochromatin. In human–human hybrids derived from the fusion of the HHV-8-carrying BCBL-1 cells and the EBV-immortalized lymphoblastoid cell line IB4, LNA-1 did not co-localize with EBNA-1, EBNA-2, EBNA-5 or EBNA-6. LNA-1 was not associated with PML containing ND10 bodies either. DNase but not RNase or detergent treatment of isolated nuclei destroys LNA-1 bodies. In advanced apoptotic cells LNA- 1 bodies remain intact but are not included in the apoptotic bodies themselves.
Porcine adenovirus-3 as a helper-dependent expression vector
Authors: Reddy P. Seshidhar , Idamakanti Neeraja , Babiuk Lorne A. , Mehtali Majid , Tikoo Suresh K.
Published: August 2015

Porcine adenovirus has been proposed as a potential vector for generating novel and effective vaccines for pigs. As a prerequisite for the generation of helper-dependent porcine adenovirus-3 (PAV-3) vectors, two E1-complementing porcine cell lines expressing E1 proteins of human adenovirus-5 (HAV-5) were made. These cell lines could be efficiently transfected with DNA and allowed the rescue and propagation of a PAV-3 recombinant, PAV201, containing a 0·597 kb E3 deletion and a 0·803 kb E1A deletion. Our data demonstrate that E1A proteins of HAV-5 have the capacity to transform foetal porcine retina cells and complement for the E1A proteins of PAV-3. The green fluorescent protein (GFP) gene placed under the control of a cytomegalovirus immediate early promoter was inserted into the E1A region of the PAV201 genome. Using these cell lines, a helper-dependent PAV-3 recombinant expressing GFP, PAV202, was constructed and characterized. The wild-type PAV-3 and the recombinant PAV202 expressing GFP were used to determine the ability of the virus to enter and replicate in cells of human and animal origin under cell culture conditions. Our results suggest that PAV-3 enters but does not replicate in dog, sheep, bovine and human cells.
Lack of negative influence on the cellular transcription factors NF-κB and AP-1 by the Nef protein of human immunodeficiency virus type 1
Authors: Yoon Keejung , Kim Sunyoung
Published: August 2015

In order to investigate the molecular mechanism of the reported negative effect of the Nef protein of human immunodeficiency virus type 1 (HIV-1) on the cellular transcription factors NF-κB and AP-1, human T cell lines (both populations and subclones) expressing the nef gene from HIV-1 clone pNL432 were constructed. Functional expression of the nef gene was confirmed by downregulation of CD4 and MHC class I proteins on the cell surface as measured by fluorescence-activated cell sorter analysis. However, contrary to previous reports, no significant difference was found in the induced level of NF-κB and AP-1 activity between nef
+ and nef
− cell lines upon stimulation by phorbol 12-myristate 13-acetate and phytohaemagglutinin, as measured by transient transfection and electromobility shift assays. These data indicate that the Nef protein does not have a negative effect on the induction of NF-κB and AP-1.
Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA
Authors: Hurrelbrink Robert J. , Nestorowicz Ann , McMinn Peter C.
Published: August 2015

An infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was constructed by stably inserting genome-length cDNA into the low-copy-number plasmid vector pMC18. Designated pMVE-1-51, the clone consisted of genome-length cDNA of MVE-1-51 under the control of a T7 RNA polymerase promoter. The clone was constructed by using existing components of a cDNA library, in addition to cDNA of the 3′ terminus derived by RT–PCR of poly(A)-tailed viral RNA. Upon comparison with other flavivirus sequences, the previously undetermined sequence of the 3′ UTR was found to contain elements conserved throughout the genus Flavivirus. RNA transcribed from pMVE-1-51 and subsequently transfected into BHK-21 cells generated infectious virus. The plaque morphology, replication kinetics and antigenic profile of clone-derived virus (CDV-1-51) was similar to the parental virus in vitro. Furthermore, the virulence properties of CDV-1-51 and MVE-1-51 (LD50 values and mortality profiles) were found to be identical in vivo in the mouse model. Through site-directed mutagenesis, the infectious clone should serve as a valuable tool for investigating the molecular determinants of virulence in MVE virus.
Vaccination of cattle with a DNA plasmid encoding the bovine viral diarrhoea virus major glycoprotein E2
Authors: Harpin Serge , Hurley David J. , Mbikay Majambu , Talbot Brian , Elazhary Youssef
Published: August 2015

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle that is ubiquitously distributed worldwide. In this study, cattle were immunized by intramuscular injections with plasmid DNA expressing the BVDV type 1 major glycoprotein E2. Animals either received injections of naked DNA (N-DNA) or DNA in cationic liposomes (L-DNA). Both DNA preparations induced virus-specific neutralizing antibodies in vaccinates, although the response was much lower in N-DNA-immunized animals. N-DNA-vaccinated animals also showed virus-specific lymphocyte proliferation responses to type 1, live BVDVin vitro, whereas L-DNA vaccination induced no such responses. After 16 weeks, DNA-vaccinated and mock-vaccinated animals were challenged with a USDA-certified BVDV type 1 strain. Four significant observations were made: (1) N-DNA-vaccinated calves showed limited protection from virus challenge, (2) L-DNA-vaccinated animals did not show any signs of protection, (3) the challenge induced strong memory responses in the production of serum neutralizing antibodies to both genotypes (type 1 and 2 of BVDV), and (4) the challenge induced a mucosal memory response in nasal secretions of both L- and N-DNA-vaccinated animals.
Comparative analysis of virus–host cell interactions of haemagglutinating and non-haemagglutinating strains of coxsackievirus B3
Authors: Pasch Andreas , Küpper Jan-Heiner , Wolde Antje , Kandolf Reinhard , Selinka Hans-Christoph
Published: August 2015

Decay-accelerating factor (DAF/CD55), and coxsackievirus–adenovirus receptor (CAR) have been identified as cellular receptors for coxsackie B viruses (CBV). To elucidate the interplay of DAF and CAR on the cell surface, virus–receptor interactions of two coxsackieviruses of serotype B3 (non-haemagglutinating CBV3 and haemagglutinating CBV3-HA strain) were analysed. Binding assays revealed clear differences between these viruses with regard to their interactions with DAF and CAR. However, only the combination of anti-DAF and anti-CAR antibodies resulted in complete inhibition of virus binding for both strains. In plaque-reduction assays, anti-DAF antibodies had no effect, whereas CAR-specific antibodies significantly reduced productive infection of HeLa cells by both viruses. Interestingly, a synergistic inhibitory effect of anti-DAF and anti-CAR antibodies was also observed with regard to infection. These findings support the model of preferential interactions of both strains of CBV3 with closely associated DAF and CAR proteins on HeLa cells, despite displaying clear differences in their binding phenotypes.
Expression in cattle of epitopes of a heterologous virus using a recombinant rinderpest virus
Authors: Baron Michael D. , Foster-Cuevas Mildred , Baron Jana , Barrett Thomas
Published: August 2015

We have investigated the bovine immune response to heterologous proteins expressed using a recombinant rinderpest virus (RPV). A new gene unit was created in a cDNA copy of the genome of the vaccine strain of RPV, and an open reading frame inserted that encodes the polymerase (3Dpol) and parts of the capsid protein VP1 from foot-and-mouth disease virus (FMDV). Infectious recombinant RPV was rescued and shown to express the FMDV-derived protein at good levels in infected cells. The rescued virus was only slightly more attenuated in tissue culture than the original virus. Cattle infected with this recombinant generated a normal immune response to RPV, and were protected from lethal challenge by that virus. Experimental animals showed a specific delayed-type hypersensitivity response to FMDV 3Dpol, similar to that seen in FMDV infection; however, no antibodies were detected recognizing either of the components of the FMDV-derived protein, nor was any proliferative response to these epitopes found in isolated peripheral blood lymphocytes from infected animals. No protection was seen against FMDV infection.
Cellular transcription factors regulate human papillomavirus type 16 gene expression by binding to a subset of the DNA sequences recognized by the viral E2 protein
Authors: Lewis Hannah , Webster Kenneth , Sanchez-Perez Ana-Maria , Gaston Kevin
Published: August 2015

Human papillomavirus type 16 (HPV-16) is a DNA tumour virus that has been implicated in the development of cervical cancer. The HPV-16 E2 protein binds to four sites that are present upstream of the viral P97 promoter and regulates transcription of the E6 and E7 oncogenes. Here, it is shown that cellular transcription factors bind to two of these E2 sites. One cellular E2 site-binding factor, which is here named CEF-1, binds tightly to E2 site 1. CEF-2, an unrelated cellular E2 site-binding factor, binds tightly to E2 site 3. Transient transfection studies performed in the absence of the E2 protein showed that mutations that blocked the binding of CEF-1 to E2 site 1 or CEF-2 to E2 site 3 significantly reduced P97 promoter activity. Further characterization of CEF-1 indicated that this factor has not previously been identified and that CEF-1 and E2 competed for binding at E2 site 1.
Regulation of cyclin E gene expression by the human papillomavirus type 16 E7 oncoprotein
Authors: Vogt Beate , Zerfaß-Thome Karin , Schulze Almut , Botz Jürgen W. , Zwerschke Werner , Jansen-Dürr Pidder
Published: August 2015

In this study, we characterized the 5′ regulatory region of the murine cyclin E gene and analysed activation of the gene by the E7 oncogene of human papillomavirus type 16 in transfection experiments. We found that the murine cyclin E promoter is composed of multiple regulatory elements, and we present evidence for at least two independent transcription units, designated P1 and P2. Overlapping binding sites for the cellular transcription factors Sp1 and E2F were identified in both promoters, and we found that E2F-mediated activation of transcription is inhibited by Sp1 in cotransfection experiments. The E2F/Sp1 binding sites contribute to transcriptional activation by E7, and the data suggest that the cyclin E gene is rendered E7-inducible through the combination of severalcis-acting elements which display only weak intrinsic responsiveness to E7.
The vaccinia virus A40R gene product is a nonstructural, type II membrane glycoprotein that is expressed at the cell surface
Authors: Wilcock Diane , Duncan Stephen A. , Traktman Paula , Zhang Wei-Hong , Smith Geoffrey L.
Published: August 2015

Gene A40R from vaccinia virus (VV) strain Western Reserve has been characterized. The open reading frame (ORF) was predicted to encode a 159 amino acid, 18152 Da protein with amino acid similarity to C-type animal lectins and to the VV A34R protein, a component of extracellular enveloped virus (EEV). Northern blotting and S1 nuclease mapping showed that gene A40R is transcribed early during infection from a position 12 nucleotides upstream of the ORF, producing a transcript of approximately 600 nucleotides. Rabbit anti-sera were raised against bacterial fusion proteins containing parts of the A40R protein. These were used to identify an 18 kDa primary translation product andN- andO-glycosylated forms of 28, 35 and 38 kDa. The A40R proteins were detected early during infection, formed higher molecular mass complexes under non-reducing conditions and were present on the cell surface but absent from virions. The proteins partitioned with integral membrane proteins in Triton X-114. Canine pancreatic microsomal membranes protectedin vitro-translated A40R from proteinase K digestion, suggesting the A40R protein has type II membrane topology. A mutant virus with the A40R gene disrupted after amino acid 50, so as to remove the entire lectin-like domain, and a revertant virus were constructed. Disruption of the A40R gene did not affect virus plaque size,in vitrogrowth rate and titre, EEV formation, or virus virulence in a murine intranasal model.
Functional features of the bovine enterovirus 5′-non-translated region
Authors: Zell Roland , Sidigi Karim , Henke Andreas , Schmidt-Brauns Joachim , Hoey Elizabeth , Martin Sam , Stelzner Axel
Published: August 2015

The bovine enterovirus (BEV) serotypes exhibit unique features of the non-translated regions (NTRs) which separate them from the other enteroviruses. Their most remarkable property is an additional genome region of 110 nt located between the 5′-cloverleaf and the internal ribosome entry site (IRES). This genome region has the potential to form an additional cloverleaf structure (domain I*) separated from the 5′-cloverleaf (domain I) by a small stem–loop (domain I**). Other characteristics involve the putative IRES domains III and VI. In order to investigate the features of the 5′-NTR, several full-length coxsackievirus B3 (CVB3) cDNA plasmids with hybrid 5′-NTRs were engineered. After exchange of the CVB3 cloverleaf with the BEV1 genome region representing both cloverleafs, a viable virus chimera was generated. Deletion of domain I** within the exchanged region also yielded viable virus albeit with reduced growth capacity. Deletion of sequences encoding either the first or the second BEV cloverleaf resulted in non-infectious constructs. Hybrid plasmids with exchanges of the IRES-encoding sequence or the complete 5′-NTR were non-infectious. Transfection experiments with SP6 transcripts containing 5′-NTRs fused to the luciferase message indicated that IRES-driven translation is enhanced by the presence of the CVB3 cloverleaf and both BEV1 cloverleaf structures, respectively. Deletion of either the first or the second BEV cloverleaf domain reduced but did not abolish enhanced luciferase expression. These results suggest that the substitution of two putative BEV cloverleaf structures for the putative coxsackieviral cloverleaf yields viable virus, while BEV sequences encoding the IRES fail to functionally replace CVB3 IRES-encoding sequences.
Replication of equine herpesvirus type 1 in freshly isolated equine peripheral blood mononuclear cells and changes in susceptibility following mitogen stimulation
Authors: van der Meulen Karen M. , Nauwynck Hans J. , Buddaert Wim , Pensaert Maurice B.
Published: August 2015

In the present study, the outcome of an inoculation of equine peripheral blood mononuclear cells (PBMC) with equine herpesvirus type 1 (EHV-1) was studiedin vitro. Cytoplasmic and plasma membrane expression of viral antigens, intra- and extracellular virus titres, and plaque formation in co-culture were determined. EHV-1 replicated in monocytes, although in a highly restricted way. Viral antigens were found at maximum levels (8·7% of the monocytes) at 12 h post-infection. The infection was productive in 0·16% of the monocytes. The virus yield was 100·7TCID50per productive cell. In a population of resting lymphocytes, 0·9% of cells were infected and less than 0·05% produced infectious virus. After prestimulation with different mitogens, the number of infected lymphocytes increased four to twelve times. The susceptible lymphocytes were T-lymphocytes. In mitogen-stimulated lymphocytes, clear expression of viral antigens was found on the plasma membrane.
Phylogenetic analysis of some large double-stranded RNA replicons from plants suggests they evolved from a defective single-stranded RNA virus
Authors: Gibbs Mark J. , Koga Ryuichi , Moriyama Hiromitsu , Pfeiffer Pierre , Fukuhara Toshiyuki
Published: August 2015

Sequences were recently obtained from four double-stranded (ds) RNAs from different plant species. These dsRNAs are not associated with particles and as they appeared not to be horizontally transmitted, they were thought to be a kind of RNA plasmid. Here we report that the RNA-dependent RNA polymerase (RdRp) and helicase domains encoded by these dsRNAs are related to those of viruses of the alpha-like virus supergroup. Recent work on the RdRp sequences of alpha-like viruses raised doubts about their relatedness, but our analyses confirm that almost all the viruses previously assigned to the supergroup are related. Alpha-like viruses have single-stranded (ss) RNA genomes and produce particles, and they are much more diverse than the dsRNAs. This difference in diversity suggests the ssRNA alpha-like virus form is older, and we speculate that the transformation to a dsRNA form began when an ancestral ssRNA virus lost its virion protein gene. The phylogeny of the dsRNAs indicates this transformation was not recent and features of the dsRNA genome structure and translation strategy suggest it is now irreversible. Our analyses also show some dsRNAs from distantly related plants are closely related, indicating they have not strictly co-speciated with their hosts. In view of the affinities of the dsRNAs, we believe they should be classified as viruses and we suggest they be recognized as members of a new virus genus (Endornavirus) and family (Endoviridae).
Infection with an H2 recombinant herpes simplex virus vector results in expression of MHC class I antigens on the surfaces of human neuroblastoma cells in vitro and mouse sensory neurons in vivo
Authors: Abendroth Allison , Simmons Anthony , Efstathiou Stacey , Pereira Rosemarie A.
Published: August 2015

The majority of neurons in herpes simplex virus (HSV)-infected murine sensory ganglia are transiently induced to express MHC-I antigens at the cell surface, whereas only a minority are themselves productively infected. The aim of the current work was to determine whether MHC-I antigens can be expressed on the surfaces of infected neurons in addition to their uninfected neighbours. To address this aim a recombinant HSV type 1 strain, S-130, was used to deliver a mouse H2Kdgene, under control of the HCMV IE-1 promoter/enhancer, into human neuroblastoma cellsin vitroand mouse primary sensory neuronsin vivo. S-130 expressed H2Kdantigens on the surfaces of IMR-32 cells, a human neuroblastoma cell line that expresses very low levels of MHC-I constitutively. In K562 cells, which do not express MHC-I constitutively, H2Kdand β2-microglobulin (β2m) were shown to be co-expressed at the cell surface following S-130 infection. This observation was taken as evidence that class I heavy chain (αC) molecules encoded by the expression cassette in the HSV genome were transported to the cell surface as stable complexes with β2m. Significantly, after introduction of S-130 into flank skin, H2Kdantigens were detected on the surfaces of primary sensory neurons in ganglia innervating the inoculation site. Our data show that HSV-infected murine primary sensory neurons and human neuroblastoma cells are capable of expressing cell-surface MHC-I molecules encoded by a transgene. From this, we infer that up-regulation of αC expression is, in principle, sufficient to overcome potential impediments to neuronal cell surface expression of MHC-I complexes.
Characterization of the ectromelia virus serpin, SPI-2
Authors: Turner Stephen J. , Silke John , Kenshole Bronwyn , Ruby Janet
Published: August 2015

Poxviruses encode multiple proteins that enable them to evade host responses. Among these are serine protease inhibitors (serpins). One of the earliest serpins described, cowpox virus crmA, acts to inhibit inflammation and apoptosis. crmA homologous serpins, known as SPI-2, are conserved in rabbitpox, vaccinia and variola viruses. Here, we describe the characterization of ectromelia virus (EV) SPI-2. EV SPI-2 encodes a protein of approximately 38 kDa showing >94% identity with other poxviral homologues. Conservative changes in amino acid sequence were found within the reactive site loop and the serpin backbone. Like crmA, transient expression of SPI-2 protected cells from tumour necrosis factor-mediated apoptosis and inhibited the activity of caspases-1 and -8 but not caspases-3, -6 or granzyme B. Overall, this study demonstrates that EV SPI-2 is functionally similar to crmA, based onin vitroassays.
DNA sequence of frog adenovirus
Authors: Davison Andrew J. , Wright Kathleen M. , Harrach Balázs
Published: August 2015

The genome of frog adenovirus (FrAdV-1) was sequenced and found to be the smallest of all known adenovirus genomes. The sequence obtained was 26163 bp in size and contains a substantial direct repeat near the right terminus, implying that it was derived by recombination from a parental genome of only 25517 bp. The closest relative of FrAdV-1 proved to be turkey adenovirus 3, an avian adenovirus with no previously known near relative. Sequence comparisons showed that the two viruses have equivalent gene complements, including one gene the product of which is related to sialidases. Phylogenetic analyses supported the establishment of a fourth adenovirus genus containing these two viruses, in addition to the established genera Mastadenovirus and Aviadenovirus and the proposed genus Atadenovirus. Sixteen genes were identified as being conserved between these four lineages and were presumably inherited from an ancestral adenovirus.
Characterization of human influenza A (H5N1) virus infection in mice: neuro-, pneumo- and adipotropic infection
Authors: Nishimura Hidekazu , Itamura Shigeyuki , Iwasaki Takuya , Kurata Takeshi , Tashiro Masato
Published: August 2015

Mice (ddY strain, 4 weeks old) were infected intranasally with the H5N1 influenza viruses A/Hong Kong/156/97 (HK156) and A/Hong Kong/483/97 (HK483) isolated from humans. HK156 and HK483 required 200 and 5 p.f.u. of virus, respectively, to give a 50% lethal dose to the mice when the volume of inoculum was set at 10 μl. Both viruses caused encephalitis and severe bronchopneumonia in infected mice. The severity of lung lesions caused by the viruses was essentially similar, whereas HK483 caused more extensive lesions in the brain than did HK156. This was supported by the results of virus titration of organ homogenates, which showed that the virus titres in brains of HK483-infected mice were more than 100-fold higher than those of HK156-infected mice, while those in lungs were almost equivalent. Both viruses were detected in homogenates of the heart, liver, spleen and kidney and blood of the infected mice. Virus antigen was detected by immunohistology in the heart and liver, albeit sporadically, but caused no degenerative change in these organs. The antigen was not detected in the thymus, spleen, pancreas, kidney or gastrointestinal tract. In contrast, virus antigen was found frequently in adipose tissues attached to those organs. The adipose tissues showed severe degenerative change and the virus titres in the tissues were high and comparable to those in lungs. Thus, infection of HK156 and HK483 in our mouse model was pneumo-, neuro- and adipotropic, but not pantropic. Furthermore, HK483 showed higher neurotropism than HK156, which may account for its higher lethality.
Expression of unglycosylated mutated prion protein facilitates PrPSc formation in neuroblastoma cells infected with different prion strains
Authors: Korth Carsten , Kaneko Kiyotoshi , Prusiner Stanley B.
Published: August 2015

Prion replication involves conversion of the normal, host-encoded prion protein PrPC, which is a sialoglycoprotein bound to the plasma membrane by a glycophosphatidylinositol anchor, into a pathogenic isoform, PrPSc. In earlier studies, tunicamycin prevented glycosylation of PrPC in scrapie-infected mouse neuroblastoma (ScN2a) cells but it was still expressed on the cell surface and converted into PrPSc; mutation of PrPC at glycosylation consensus sites (T182A, T198A) produced low steady-state levels of PrP that were insufficient to propagate prions in transgenic mice. By mutating asparagines to glutamines at the consensus sites, we obtained expression of unglycosylated, epitope-tagged MHM2PrP(N180Q,N196Q), which was converted into PrPSc in ScN2a cells. Cultures of uninfected neuroblastoma (N2a) cells transiently expressing mutated PrP were exposed to brain homogenates prepared from mice infected with the RML, Me7 or 301V prion strains. In each case, mutated PrP was converted into PrPSc as judged by Western blotting. These findings raise the possibility that the N2a cell line can support replication of different strains of prions.
Murine gammaherpesvirus-68 infection of and persistence in the central nervous system
Authors: Terry Linda A. , Stewart James P. , Nash Anthony A. , Fazakerley John K.
Published: August 2015

Murine gammaherpesvirus-68 (MHV-68) was originally isolated from a bank vole by passage through mouse brain. Given its ability to replicate in mouse brain and its subsequent reisolation from trigeminal ganglia, it was originally considered to be an alphaherpesvirus. Molecular studies have now firmly established MHV-68 to be a gammaherpesvirus. Other gammaherpesviruses have been suggested to cause and in some cases shown to cause neurological disease. Given the isolation history of MHV-68, we have studied the ability of this virus to gain access to, to replicate in and to persist in the mouse CNS. Following intranasal inoculation the virus was not generally neuroinvasive. However, in mice with a deletion of the type-I interferon receptor gene, peripheral virus titres are higher and perivascular CNS infection was observed. There was no evidence of virus spread via olfactory routes. Direct intracerebral inoculation of virus was fatal with widespread infection and destruction predominantly of meningeal and ependymal cells. Hippocampal pyramidal neurons, oligodendrocytes, Bergmann glia cells in the cerebellar cortex and neural progenitor cells in the rostral migratory stream were also infected. A similar infection was observed in younger mice. CNS infection following virus reactivation was investigated by implantation of infected glial cells. Implantation into a brain ventricle led to widespread fatal infection, principally involving ependymal and meningeal cells. Implantation into the striatum resulted in a predominantly neuronal infection. Implantation of cells into mice transiently treated with the antiviral thionucleoside analogue 2′-deoxy-5-ethyl-β-4′-thiouridine resulted in survival with detection of virus-infected cells in the brain 1 year later.
Heterologous protection against lethal A/HongKong/156/97 (H5N1) influenza virus infection in C57BL/6 mice
Authors: O’Neill Eduardo , Krauss Scott L. , Riberdy Janice M. , Webster Robert G. , Woodland David L.
Published: August 2015

The continual threat posed by newly emerging influenza virus strains is demonstrated by the recent outbreak of H5N1 influenza virus in Hong Kong. Currently, immunization against influenza virus infection is fairly adequate, but it is imperative that improved vaccines are developed that can protect against a variety of strains and be generated rapidly. Since humoral immunity is ineffective against serologically distinct viruses, one strategy would be to develop vaccines that emphasize cellular immunity. Here we report the successful protection of C57BL/6 mice from a lethal A/HK/156/97 (HK156) infection by immunizing first with an H9N2 isolate, A/Quail/HK/G1/97 (QHKG1), that harbours internal genes 98% homologous to HK156. This strategy also protected mice that are deficient in antibody production, indicating that the immunity is T-cell-mediated. In the course of these studies, we generated a highly pathogenic H5N1 reassortant which implicated NP and PB2 as having an important contribution to pathogenesis when present with a highly cleavable H5. These results provide the first demonstration that protective cell-mediated immunity can be established against the highly virulent HK156 virus and have important implications for the development of novel strategies for the prevention and treatment of HK156 infection and the design of future influenza vaccines.
Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro
Authors: Wang Aiming , Sanfaçon Hélène
Published: August 2015

Tomato ringspot nepovirus RNA-1-encoded polyprotein (P1) contains the domains for the putative NTP-binding protein, VPg, 3C-like protease and a putative RNA-dependent RNA polymerase in its C-terminal region. The N-terminal region of P1, with a coding capacity for a protein (or a precursor) of 67 kDa, has not been characterized. Using partial cDNA clones, it is shown that the 3C-like protease can process the N-terminal region of P1 at a novel cleavage site in vitro, allowing the release of two proteins, X1 (located at the N terminus of P1) and X2 (located immediately upstream of the NTB domain). P1 precursors in which the protease was inactive or absent were not cleaved by exogenously added protease, suggesting that P1 processing was predominantly in cis. Results from site-directed mutagenesis of putative cleavage sites suggest that dipeptides Q423/G and Q620/G are the X1-X2 and X2-NTB cleavage sites, respectively. The putative X1 protein contains a previously identified alanine-rich sequence which is present in nepoviruses but not in the related comoviruses. The putative X2 protein contains a region with similarity to the comovirus 32 kDa protease co-factor (the only mature protein released from the N terminus of comovirus P1 polyproteins) and to the corresponding region of other nepovirus P1 polyproteins. These results raise the possibility that the presence of two distinct protein domains in the N-terminal part of the P1 polyprotein may be a common feature of nepoviruses.
Mechanical transmission of Potato leafroll virus
Authors: Mayo Mike , Ryabov Eugene , Fraser Gillian , Taliansky Michael
Published: August 2015

Like typical luteoviruses, Potato leafroll virus (PLRV) cannot be transmitted mechanically by rubbing plants with solutions containing virus particles. However, PLRV was found to be mechanically transmissible from extracts of plants that had been inoculated by viruliferous aphids and then post-inoculated with Pea enation mosaic virus-2 (PEMV-2). Unlike the asymptomatic infections induced by either virus alone, double infections in Nicotiana benthamiana induced necrotic symptoms with some line patterning and vein yellowing. Infective PLRV was recovered from a purified virus preparation by inoculating plants mechanically with purified virus particles mixed with PEMV-2. Similarly, Beet mild yellowing virus was readily transmitted mechanically from mixtures containing PEMV-2. PLRV was also transmissible from mixtures made with extracts of plants infected with Groundnut rosette virus, although less efficiently than from mixtures containing PEMV-2. This novel means of transmitting PLRV, and perhaps other poleroviruses, should prove very useful in a number of fields of luteovirus research.