bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism and calculation of the niche effect in airborne sound transmission
Authors: Vinokur Roman
Published: March 2006

The goal is to interpret and calculate the “niche effect” for the airborne sound transmission through a specimen mounted inside an aperture in the wall between the source and receiving reverberation rooms. The low-frequency sound insulation is known to be worse for the specimen placed at the center than for the specimen mounted at either edge of the aperture. As shown, the aperture with a tested specimen can be simulated at low frequencies as a triple partition where the middle element is the specimen and the role of the edge leaves is played by the air masses entrained at the aperture edges. With a centrally located specimen, such a triple system is symmetric and has two main natural frequencies close together. In this case, the resonant transmission is higher than for the edge arrangement simulated as a double system with one natural frequency. Analogous resonant phenomena are known to reduce the low-frequency transmission loss for symmetric triple windows or solid walls with identical air gaps and lightweight boards on both sides. The theoretical results obtained for the mechanical and acoustical models are in a good agreement with the experimental data.
Wave propagation in two-dimensional periodic lattices
Authors: Phani A. Srikantha , Woodhouse J. , Fleck N. A.
Published: March 2006

Plane wave propagation in infinite two-dimensional periodic lattices is investigated using Floquet-Bloch principles. Frequency bandgaps and spatial filtering phenomena are examined in four representative planar lattice topologies: hexagonal honeycomb, Kagomé lattice, triangular honeycomb, and the square honeycomb. These topologies exhibit dramatic differences in their long-wavelength deformation properties. Long-wavelength asymptotes to the dispersion curves based on homogenization theory are in good agreement with the numerical results for each of the four lattices. The slenderness ratio of the constituent beams of the lattice (or relative density) has a significant influence on the band structure. The techniques developed in this work can be used to design lattices with a desired band structure. The observed spatial filtering effects due to anisotropy at high frequencies (short wavelengths) of wave propagation are consistent with the lattice symmetries.
Measuring flow resistivity of porous materials at low frequencies range via acoustic transmitted waves
Authors: Fellah Z. E. A. , Fellah M. , Sebaa N. , Lauriks W. , Depollier C.
Published: March 2006

An acoustic transmissivity method is proposed for measuring flow resistivity of porous materials having rigid frame. Flow resistivity of porous material is defined as the ratio between the pressure difference across a sample and the velocity of flow of air through that sample per unit cube. The proposed method is based on a temporal model of the direct and inverse scattering problem for the diffusion of transient low-frequency waves in a homogeneous isotropic slab of porous material having a rigid frame. The transmission scattering operator for a slab of porous material is derived from the response of the medium to an incident acoustic pulse. The flow resistivity is determined from the solution of the inverse problem. The minimization between experiment and theory is made in the time domain. Tests are performed using industrial plastic foams. Experimental and numerical results, and prospects are discussed.
Automatic determination of the number of targets present when using the time reversal operator
Authors: Quinlan Angela , Barbot Jean-Pierre , Larzabal Pascal
Published: March 2006

Acoustical time reversal mirrors have been shown to provide a highly accurate means of studying and focusing on acoustical sources. The DORT method is a derivation of the time reversal process, which allows for focusing on multiple targets. An important step in this process is the determination of the number of targets or sources present. This is achieved by examining the eigenvalues of the time reversal operator (TRO). The number of significant eigenvalues is then chosen as the number of sources present. However, as mentioned in [N. Mordant, C. Prada, and M. Fink, J. Acoust. Soc. Am. 105, 2634–2642 (1999) and C. Prada, M. Tanter, and M. Fink, in Proceedings of the IEEE Symposium, 1997, pp. 679–683], factors such as low signal to noise ratio (SNR), small data sample, array configuration and the target location may result in the eigenvalues corresponding to the targets no longer being distinguishable from the background noise eigenvalues. This paper proposes a robust method of automatically determining the number of targets even in the presence of a small number of snapshots. For white Gaussian noise, the profile of the ordered eigenvalues is seen to fit an exponential law. The observed eigenvalues are then compared to this model and a mismatch is detected between the observed profile and the noise-only model. The index of the mismatch gives the number of scatterers present.
Scattering of Rayleigh-Lamb waves by a surface breaking crack in an elastic plate
Authors: Flores-López Miguel A. , Douglas Gregory R.
Published: March 2006

This work studies the problem of scattering of Rayleigh-Lamb waves by a thin surface-breaking crack. The problem is solved by the projection method, which exploits some orthogonality relations deduced from the reciprocal theorem. Numerical results obtained by the projection method are compared with the results obtained using a finite element method reported by other authors for the case of a flat crack, finding good agreement. From the numerical results it is observed that, as the wave number of the incident wave, S0 for instance, is smaller, the reflected and transmitted energy corresponding to the converted mode, A0 in this case, are almost equal to each other. An explanation for this result is given, based on the asymptotic nature of the modes S0 and A0 for small wave numbers.
Effect of masker type on native and non-native consonant perception in noise
Authors: Lecumberri M. L. Garcia , Cooke Martin
Published: March 2006

Spoken communication in a non-native language is especially difficult in the presence of noise. This study compared English and Spanish listeners' perceptions of English intervocalic consonants as a function of masker type. Three maskers (stationary noise, multitalker babble, and competing speech) provided varying amounts of energetic and informational masking. Competing English and Spanish speech maskers were used to examine the effect of masker language. Non-native performance fell short of that of native listeners in quiet, but a larger performance differential was found for all masking conditions. Both groups performed better in competing speech than in stationary noise, and both suffered most in babble. Since babble is a less effective energetic masker than stationary noise, these results suggest that non-native listeners are more adversely affected by both energetic and informational masking. A strong correlation was found between non-native performance in quiet and degree of deterioration in noise, suggesting that non-native phonetic category learning can be fragile. A small effect of language background was evident: English listeners performed better when the competing speech was Spanish.
Automated pure-tone threshold estimations from extrapolated distortion product otoacoustic emission (DPOAE) input∕output functions
Authors: Schmuziger Nicolas , Patscheke Jochen , Probst Rudolf
Published: March 2006

A promising approach to the prediction of pure-tone thresholds through the estimation of DPOAE thresholds by input∕output functions was recently published by Boege and Janssen [J. Acoust. Soc. Am. 111, 1810–1818 (2002)]. On the basis of their results, a device that enables automated measurements of these thresholds was recently developed. The purpose of the current study was to evaluate the reliability of this instrument for the objective assessment of hearing loss in 101 ears with either normal hearing or with cochlear hearing loss of up to 50dB HL. The median difference between pure-tone hearing and DPOAE thresholds was approximately 2dB. For individual subjects, however, DPOAE thresholds differed from pure-tone thresholds by up to 40dB. We find, therefore, that the clinical benefits of this method are probably limited.
Using a scanning laser Doppler vibrometer to measure acoustic intensity
Authors: Boone Andrew J. , Blotter Jonathan , Sommerfeldt Scott D. , Leishman Timothy W.
Published: March 2006

Two different scanning methods were compared to see if acoustic intensity can be accurately measured using a scanning laser Doppler vibrometer (SLDV). Both intensity scans were performed on the same vibrating object, the side panel of a diesel generator enclosure. The first scan was done the traditional way using an intensity probe and scanning grid. The second scan was done using an SLDV. The results from each intensity scan were compared and analyzed. From these results it appears that the SLDV scan is measuring intensity contributed by the vibrating panel alone, while the traditional scan is measuring intensity contributions from both the vibrating panel and the enclosed generator.
What is boiling during high-intensity focused ultrasound
Authors: Kreider Wayne , Bailey Michael R. , Crum Lawrence A.
Published: March 2006

For treatments that use high-intensity focused ultrasound (HIFU), it is important to understand the behavior of bubbles in the context of both large acoustic pressures and elevated temperatures in the surrounding medium. Based upon clinical and experimental observations, any preexisting cavitation nuclei in tissue or blood are likely to be less than 1 micron. For HIFU conditions characterized by megahertz frequencies and pressures on the order of megaPascals, gas bubbles less than a micron in radius can grow explosively. Calculations for a single, spherical bubble imply that the resulting bubble motions are significantly influenced by evaporation and condensation processes. Consequently, at both high and low ambient temperatures, HIFU-driven bubbles may best be described as gas-vapor bubbles that can exhibit rectified transfer of both heat and noncondensable gases. Moreover, increased vapor pressures associated with ambient temperatures at or above ‘‘boiling’’ may not lead to unbounded bubble growth as expected for a quasistatic bubble in a superheated medium. Instead, calculations suggest that growth of boiling bubbles can be confined. [Work supported by NIH T32 EB001650, NIH DK43881, and NSBRI SMS00402.]
Induction of G
2
Arrest and Binding to Cyclophilin A Are Independent Phenotypes of Human Immunodeficiency Virus Type 1 Vpr
Authors: Ardon Orly , Zimmerman Erik S. , Andersen Joshua L. , DeHart Jason L. , Blackett Jana , Planelles Vicente
Published: March 2006

Cyclophilin A (CypA) is a member of a family of cellular proteins that share a peptidyl prolyl
cis-trans
isomerase (PPIase) activity. CypA was previously reported to be required for the biochemical stability and function (specifically, induction of G
2
arrest) of the human immunodeficiency virus type 1 (HIV-1) protein R (Vpr). In the present study, we examine the role of the Vpr-CypA interaction on Vpr-induced G
2
arrest. We find that Vpr coimmunoprecipitates with CypA and that this interaction is disrupted by substitution of proline-35 of Vpr as well as incubation with the CypA inhibitor cyclosporine A (CsA). Surprisingly, the presence of CypA or its binding to Vpr is dispensable for the ability of Vpr to induce G
2
arrest. Vpr expression in CypA
−/−
cells leads to induction of G
2
arrest in a manner that is indistinguishable from that in CypA
+
cells. CsA abolished CypA-Vpr binding but had no effect on induction of G
2
arrest or Vpr steady-state levels. In view of these results, we propose that the interaction with CypA is independent of the ability of Vpr to induce cell cycle arrest. The interaction between Vpr and CypA is intriguing, and further studies should examine its potential effects on other functions of Vpr.
Systemic Immunization with an ALVAC-HIV-1/Protein Boost Vaccine Strategy Protects Rhesus Macaques from CD4+T-Cell Loss and Reduces both Systemic and Mucosal Simian-Human Immunodeficiency Virus SHIVKU2RNA Levels
Authors: Pal Ranajit , Venzon David , Santra Sampa , Kalyanaraman Vaniambadi S. , Montefiori David C. , Hocker Lindsey , Hudacik Lauren , Rose Nicolas , Nacsa Janos , Edghill-Smith Yvette , Moniuszko Marcin , Hel Zdeněk , Belyakov Igor M. , Berzofsky Jay A. , Parks Robyn Washington , Markham Phillip D. , Letvin Norman L. , Tartaglia Jim , Franchini Genoveffa
Published: March 2006

Transmission of human immunodeficiency virus type 1 (HIV-1) occurs primarily via the mucosal route, suggesting that HIV-1 vaccines may need to elicit mucosal immune responses. Here, we investigated the immunogenicity and relative efficacy of systemic immunization with two human ALVAC-HIV-1 recombinant vaccines expressing Gag, Pol, and gp120 (vCP250) or Gag, Pol, and gp160 (vCP1420) in a prime-boost protocol with their homologous vaccine native Env proteins. The relative efficacy was measured against a high-dose mucosal exposure to the pathogenic neutralization-resistant variant SHIVKU2(simian-human immunodeficiency virus). Systemic immunization with both vaccine regimens decreased viral load levels not only in blood but unexpectedly also in mucosal sites and protected macaques from peripheral CD4+T-cell loss. This protective effect was stronger when the gp120 antigen was included in the vaccine. Inclusion of recombinant Tat protein in the boosting phase along with the Env protein did not contribute further to the preservation of CD4+T cells. Thus, systemic immunization with ALVAC-HIV-1 vaccine candidates elicits anti-HIV-1 immune responses able to contain virus replication also at mucosal sites in macaques.
Vpu and Tsg101 Regulate Intracellular Targeting of the Human Immunodeficiency Virus Type 1 Core Protein Precursor Pr55

gag
Authors: Harila Kirsi , Prior Ian , Sjöberg Mathilda , Salminen Antti , Hinkula Jorma , Suomalainen Maarit
Published: March 2006

Assembly of human immunodeficiency virus type 1 (HIV-1) is directed by the viral core protein Pr55

gag

. Depending on the cell type, Pr55

gag

accumulates either at the plasma membrane or on late endosomes/multivesicular bodies. Intracellular localization of Pr55

gag

determines the site of virus assembly, but molecular mechanisms that define cell surface or endosomal targeting of Pr55

gag

are poorly characterized. We have analyzed targeting of newly synthesized Pr55

gag

in HeLa H1 cells by pulse-chase studies and subcellular fractionations. Our results indicated that Pr55

gag

was inserted into the plasma membrane and, when coexpressed with the viral accessory protein Vpu, Pr55

gag

remained at the plasma membrane and virions assembled at this site. In contrast, Pr55

gag

expressed in the absence of Vpu was initially inserted into the plasma membrane, but subsequently endocytosed, and virus assembly was partially shifted to internal membranes. This endocytosis of Pr55

gag

required the host protein Tsg101. These results identified a previously unknown role for Vpu and Tsg101 as regulators for the endocytic uptake of Pr55

gag

and suggested that the site of HIV-1 assembly is determined by factors that regulate the endocytosis of Pr55

gag

.
Stable Association of Herpes Simplex Virus with Target Membranes Is Triggered by Low pH in the Presence of the gD Receptor, HVEM
Authors: Whitbeck J. Charles , Zuo Yi , Milne Richard S. B. , Cohen Gary H. , Eisenberg Roselyn J.
Published: March 2006

Using a liposome-binding assay, we investigated the requirements for activation of herpes simplex virus (HSV) into a state capable of membrane interaction. Virions were mixed with liposomes along with the ectodomain of one of three gD receptors (HVEMt, nectin-1t, or nectin-2t) and incubated under different pH and temperature conditions. Virions failed to associate with liposomes in the presence of nectin-1 or nectin-2 at any temperature or pH tested. In contrast, HVEMt triggered association of HSV with liposomes at pH 5.3 or 5.0 when incubated at 37°C, suggesting that HVEM binding and mildly acidic pH at a physiological temperature provide coactivation signals, allowing virus association with membranes. Virions incubated with HVEMt at 37°C without liposomes rapidly lost infectivity upon exposure to pH 5.0, suggesting that these conditions lead to irreversible virus inactivation in the absence of target membranes. Consistent with the idea that soluble receptor molecules provide a trigger for HSV entry, HVEMt promoted virus entry into receptor-deficient CHO K1 cells. However, in B78H1 cells, HVEMt promoted virus entry with markedly lower efficiency. Interestingly, HSV entry into receptor-bearing CHO K1 cells has been shown to proceed via a pH-dependent manner, whereas HSV entry into receptor-bearing B78H1 cells is pH independent. Based on these observations, we propose that the changes triggered by HVEM and mildly acidic pH that allow liposome association are similar or identical to changes that occur during pH-dependent HSV entry.
Cap-Independent Translation Mechanism of
Red Clover Necrotic Mosaic Virus
RNA2 Differs from That of RNA1 and Is Linked to RNA Replication
Authors: Mizumoto Hiroyuki , Iwakawa Hiro-oki , Kaido Masanori , Mise Kazuyuki , Okuno Tetsuro
Published: March 2006

The genome of
Red clover necrotic mosaic virus
(RCNMV) in the genus
Dianthovirus
is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5′ end and no poly(A) tail at the 3′ end. The 3′ untranslated region (3′ UTR) of RCNMV RNA1 contains an essential RNA element (3′TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect
cis
-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3′TE-DR1 of RNA1. However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5′ UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in
cis
-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.
Identification of Functional Domains in Herpes Simplex Virus 2 Glycoprotein B
Authors: Li Wei , Minova-Foster Tanja J. , Norton Daniel D. , Muggeridge Martin I.
Published: March 2006

Glycoprotein B (gB) is one of four membrane proteins that are essential for the entry of herpes simplex viruses (HSV) into cells, and coexpression of the same combination of proteins in transfected cells results in cell fusion. The latter effect is reminiscent of the ability of virus infection to cause cell fusion, particularly since the degree of fusion is greatly increased by syncytial mutations in gB. Despite intensive efforts with the gB homologs of HSV and some other herpesviruses, information about functionally important regions in the 700-amino-acid ectodomain of this protein is very limited at present. This is largely due to the misfolding of the majority of the mutants examined. It was shown previously that the percentage of correctly folded mutants could be increased by targeting only predicted loop regions (i.e., not alpha-helix or beta-strand), and by using this approach new functional domains in HSV-2 gB have now been identified.
Evidence that the Human Cytomegalovirus IE2-86 Protein Binds mdm2 and Facilitates mdm2 Degradation
Authors: Zhang Zhigang , Evers David L. , McCarville Joseph F. , Dantonel Jean-Christophe , Huong Shu-Mei , Huang Eng-Shang
Published: March 2006

Levels of the p53 tumor suppressor protein are increased in human cytomegalovirus (HCMV)-infected cells and may be important for HCMV pathogenesis. In normal cells p53 levels are kept low due to an autoregulatory feedback loop where p53 activates the transcription of mdm2 and mdm2 binds and ubiquitinates p53, targeting p53 for proteasomal degradation. Here we report that, in contrast to uninfected cells, mdm2 was undetectable upon treatment of infected fibroblasts with the proteasome inhibitor MG132. Cellular depletion of mdm2 was reproducible in p53-null cells transfected with the HCMV IE2-86 protein, but not with IE172, independently of the endogenous mdm2 promoter. IE2-86 also prevented the emergence of presumably ubiquitinated species of p53. The regions of IE2-86 important for mdm2 depletion were those containing the sequences corresponding to the putative zinc finger and C-terminal acidic motifs. mdm2 and IE2-86 coimmunoprecipitated in transfected and infected cell lysates and in a cell-free system. IE2-86 blocked mdm2's p53-independent transactivation of the cyclin A promoter in transient-transfection experiments. Pulse-chase experiments revealed that IE2-86 but not IE1-72 or several loss-of-function IE2-86 mutants increased the half-life of p53 and reduced the half-life of mdm2. Short interfering RNA-mediated depletion of IE2-86 restored the ability of HCMV-infected cells to accumulate mdm2 in response to proteasome inhibition. Taken together, the data suggest that specific interactions between IE2-86 and mdm2 cause proteasome-independent degradation of mdm2 and that this may be important for the accumulation of p53 in HCMV-infected cells.
Characterization of Functional Domains of Equine Infectious Anemia Virus Rev Suggests a Bipartite RNA-Binding Domain
Authors: Lee Jae-Hyung , Murphy Sean C. , Belshan Michael , Sparks Wendy O. , Wannemuehler Yvonne , Liu Sijun , Hope Thomas J. , Dobbs Drena , Carpenter Susan
Published: March 2006

Equine infectious anemia virus (EIAV) Rev is an essential regulatory protein that facilitates expression of viral mRNAs encoding structural proteins and genomic RNA and regulates alternative splicing of the bicistronic
tat/rev
mRNA. EIAV Rev is characterized by a high rate of genetic variation in vivo, and changes in Rev genotype and phenotype have been shown to coincide with changes in clinical disease. To better understand how genetic variation alters Rev phenotype, we undertook deletion and mutational analyses to map functional domains and to identify specific motifs that are essential for EIAV Rev activity. All functional domains are contained within the second exon of EIAV Rev. The overall organization of domains within Rev exon 2 includes a nuclear export signal, a large central region required for RNA binding, a nonessential region, and a C-terminal region required for both nuclear localization and RNA binding. Subcellular localization of green fluorescent protein-Rev mutants indicated that basic residues within the KRRRK motif in the C-terminal region of Rev are necessary for targeting of Rev to the nucleus. Two separate regions of Rev were necessary for RNA binding: a central region encompassing residues 57 to 130 and a C-terminal region spanning residues 144 to 165. Within these regions were two distinct, short arginine-rich motifs essential for RNA binding, including an RRDRW motif in the central region and the KRRRK motif near the C terminus. These findings suggest that EIAV Rev utilizes a bipartite RNA-binding domain.
Adaptive Evolution and Antiviral Activity of the Conserved Mammalian Cytidine Deaminase
APOBEC3H
Authors: OhAinle Molly , Kerns Julie A. , Malik Harmit S. , Emerman Michael
Published: March 2006

The
APOBEC3
genes encode cytidine deaminases that act as components of an intrinsic immune defense that have potent activity against a variety of retroelements. This family of genes has undergone a rapid expansion from one or two genes in nonprimate mammals to at least seven members in primates. Here we describe the evolution and function of an uncharacterized antiviral effector,
APOBEC3H
, which represents the most evolutionarily divergent
APOBEC3
gene found in primates. We found that
APOBEC3H
has undergone significant adaptive evolution in primates. Consistent with our previous findings implicating adaptively evolving
APOBEC3
genes as antiviral effectors, APOBEC3H from Old World monkeys (OWMs) has efficient antiviral activity against primate lentiviruses, is sensitive to inactivation by the simian immunodeficiency virus Vif protein, and is capable of hypermutating retroviral genomes. In contrast, human APOBEC3H is inherently poorly expressed in primate cells and is ineffective at inhibiting retroviral replication. Both OWM and human APOBEC3H proteins can be expressed in bacteria, where they display significant DNA mutator activity. Thus, humans have retained an
APOBEC3H
gene that encodes a functional, but poorly expressed, cytidine deaminase with no apparent antiviral activity. The consequences of the lack of antiviral activity of human APOBEC3H are likely to be relevant to the current-day abilities of humans to combat retroviral challenges.
Inactivating a Cellular Intrinsic Immune Defense Mediated by Daxx Is the Mechanism through Which the Human Cytomegalovirus pp71 Protein Stimulates Viral Immediate-Early Gene Expression
Authors: Saffert Ryan T. , Kalejta Robert F.
Published: March 2006

Human cytomegalovirus (HCMV) masterfully evades adaptive and innate immune responses, allowing infection to be maintained and periodically reactivated for the life of the host. Here we show that cells also possess an intrinsic immune defense against HCMV that is disarmed by the virus. In HCMV-infected cells, the promyelocytic leukemia nuclear body (PML-NB) protein Daxx silences viral immediate-early gene expression through the action of a histone deacetylase. However, this antiviral tactic is efficiently neutralized by the viral pp71 protein, which is incorporated into virions, delivered to cells upon infection, and mediates the proteasomal degradation of Daxx. This work demonstrates the mechanism through which pp71 activates viral immediate-early gene expression in HCMV-infected cells. Furthermore, it provides insight into how a PML-NB protein institutes an intrinsic immune defense against a DNA virus and how HCMV pp71 inactivates this defense.
Inhibition of Cellular DNA Synthesis by the Human Cytomegalovirus IE86 Protein Is Necessary for Efficient Virus Replication
Authors: Petrik Dustin T. , Schmitt Kimberly P. , Stinski Mark F.
Published: March 2006

Human cytomegalovirus (HCMV) expresses several proteins that manipulate normal cellular functions, including cellular transcription, apoptosis, immune response, and cell cycle control. The IE2 gene, which is expressed from the HCMV major immediate-early (MIE) promoter, encodes the IE86 protein. IE86 is a multifunctional protein that is essential for viral replication. The functions of IE86 include transactivation of cellular and viral early genes, negative autoregulation of the MIE promoter, induction of cell cycle progression from G
0
/G
1
to G
1
/S, and arresting cell cycle progression at the G
1
/S transition in p53-positive human foreskin fibroblast (HFF) cells. Mutations were introduced into the IE2 gene in the context of the viral genome using bacterial artificial chromosomes (BACs). From these HCMV BACs, a recombinant virus (RV) with a single amino acid substitution in the IE86 protein was isolated that replicates slower and to lower titers than wild-type HCMV. HFF cells infected with the Q548R RV undergo cellular DNA synthesis and do not arrest at any point in the cell cycle. The Q548R RV is able to negatively autoregulate the MIE promoter, transactivate viral early genes, activate cellular E2F-responsive genes, and produce infectious virus. This is the first report of a viable recombinant HCMV that is unable to inhibit cellular DNA synthesis in infected HFF cells.
Characterization of the Recombinant Adenovirus Vector AdYB-1: Implications for Oncolytic Vector Development
Authors: Glockzin Gabriel , Mantwill Klaus , Jurchott Karsten , Bernshausen Alexandra , Ladhoff Axel , Royer Hans-Dieter , Gansbacher Bernd , Holm Per Sonne
Published: March 2006

Conditionally replicating adenoviruses are a promising new modality for the treatment of cancer. However, early clinical trials demonstrate that the efficacy of current vectors is limited. Interestingly, DNA replication and production of viral particles do not always correlate with virus-mediated cell lysis and virus release depending on the vector utilized for infection. However, we have previously reported that nuclear accumulation of the human transcription factor YB-1 by regulating the adenoviral E2 late promoter facilitates viral DNA replication of E1-deleted adenovirus vectors which are widely used for cancer gene therapy. Here we report the promotion of virus-mediated cell killing as a new function of the human transcription factor YB-1. In contrast to the E1A-deleted vector dl312 the first-generation adenovirus vector AdYB-1, which overexpresses YB-1 under cytomegalovirus promoter control, led to necrosis-like cell death, virus production, and viral release after infection of A549 and U2OS tumor cell lines. Our data suggest that the integration of YB-1 in oncolytic adenoviruses is a promising strategy for developing oncolytic vectors with enhanced potency against different malignancies.
Rotavirus Spike Protein VP4 Binds to and Remodels Actin Bundles of the Epithelial Brush Border into Actin Bodies
Authors: Gardet Agnès , Breton Michelyne , Fontanges Philippe , Trugnan Germain , Chwetzoff Serge
Published: March 2006

We demonstrate here that VP4, a rotaviral protein, is able to specifically bind to bundled actin microfilaments that are subsequently profoundly remodeled into actin bodies. These cytoplasmic actin bodies do not localize within identified intracellular compartments. VP4-induced actin remodeling is similar to cytochalasin D effects with kinetics compatible with that of rotavirus infection. Actin bundles' remodeling occurs both in infected and in VP4-transfected cells and in various cell lines, indicating that this is a general property of the viral protein itself. Interestingly, in intestinal epithelial cells, which represent the natural target of rotavirus, VP4 is addressed to the apical membrane where it binds specifically to brush border actin bundles and elicits its remodeling, whereas cytochalasin D impaired all the filamentous actin. These observations indicate that these original properties of VP4 likely explain the previously described brush border alterations that follow rotavirus infection of enterocytes and may also participate to the mechanism of rotavirus final assembly.
The CPSF30 Binding Site on the NS1A Protein of Influenza A Virus Is a Potential Antiviral Target
Authors: Twu Karen Y. , Noah Diana L. , Rao Ping , Kuo Rei-Lin , Krug Robert M.
Published: March 2006

The emergence of influenza A viruses resistant to the two existing classes of antiviral drugs highlights the need for additional antiviral drugs, particularly considering the potential threat of a pandemic of H5N1 influenza A viruses. Here, we determine whether influenza A virus replication can be selectively inhibited by blocking the ability of its NS1A protein to inhibit the 3′-end processing of cellular pre-mRNAs, including beta interferon (IFN-β) pre-mRNA. Pre-mRNA processing is inhibited via the binding of the NS1A protein to the cellular CPSF30 protein, and mutational inactivation of this NS1A binding site causes severe attenuation of the virus. We demonstrate that binding of CPSF30 is mediated by two of its zinc fingers, F2F3, and that the CPSF30/F2F3 binding site on the NS1A protein extends from amino acid 144 to amino acid 186. We generated MDCK cells that constitutively express epitope-tagged F2F3 in the nucleus, although at only approximately one-eighth the level of the NS1A protein produced during virus infection. Influenza A virus replication was inhibited in this cell line, whereas no inhibition was observed with influenza B virus, whose NS1B protein lacks a binding site for CPSF30. Influenza A virus, but not influenza B virus, induced increased production of IFN-β mRNA in the F2F3-expressing cells. These results, which indicate that F2F3 inhibits influenza A virus replication by blocking the binding of endogenous CPSF30 to the NS1A protein, point to this NS1A binding site as a potential target for the development of antivirals directed against influenza A virus.
Analysis of Rubella Virus Capsid Protein-Mediated Enhancement of Replicon Replication and Mutant Rescue
Authors: Tzeng Wen-Pin , Matthews Jason D. , Frey Teryl K.
Published: March 2006

The rubella virus capsid protein (C) has been shown to complement a lethal deletion (termed ΔNotI) in P150 replicase protein. To investigate this phenomenon, we generated two lines of Vero cells that stably expressed either C (C-Vero cells) or C lacking the eight N-terminal residues (CΔ8-Vero cells), a construct previously shown to be unable to complement ΔNotI. In C-Vero cells but not Vero or CΔ8-Vero cells, replication of a wild-type (wt) replicon expressing the green fluorescent protein (GFP) reporter gene (RUBrep/GFP) was enhanced, and replication of a replicon with ΔNotI (RUBrep/GFP-ΔNotI) was rescued. Surprisingly, replicons with deleterious mutations in the 5′ and 3′
cis
-acting elements were also rescued in C-Vero cells. Interestingly, the CΔ8 construct localized to the nucleus while the C construct localized in the cytoplasm, explaining the lack of enhancement and rescue in CΔ8-Vero cells since rubella virus replication occurs in the cytoplasm. Enhancement and rescue in C-Vero cells were at a basic step in the replication cycle, resulting in a substantial increase in the accumulation of replicon-specific RNAs. There was no difference in translation of the nonstructural proteins in C-Vero and Vero cells transfected with the wt and mutant replicons, demonstrating that enhancement and rescue were not due to an increase in the efficiency of translation of the transfected replicon transcripts. In replicon-transfected C-Vero cells, C and the P150 replicase protein associated by coimmunoprecipitation, suggesting that C might play a role in RNA replication, which could explain the enhancement and rescue phenomena. A unifying model that accounts for enhancement of wt replicon replication and rescue of diverse mutations by the rubella virus C protein is proposed.
Hepatitis B Surface Antigen Vector Delivers Protective Cytotoxic T-Lymphocyte Responses to Disease-Relevant Foreign Epitopes
Authors: Woo Wai-Ping , Doan Tracy , Herd Karen A. , Netter Hans-Jürgen , Tindle Robert W.
Published: March 2006

Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.
Envelope Conformational Changes Induced by Human Immunodeficiency Virus Type 1 Attachment Inhibitors Prevent CD4 Binding and Downstream Entry Events
Authors: Ho Hsu-Tso , Fan Li , Nowicka-Sans Beata , McAuliffe Brian , Li Chang-Ben , Yamanaka Gregory , Zhou Nannan , Fang Hua , Dicker Ira , Dalterio Richard , Gong Yi-Fei , Wang Tao , Yin Zhiwei , Ueda Yasutsugu , Matiskella John , Kadow John , Clapham Paul , Robinson James , Colonno Richard , Lin Pin-Fang
Published: March 2006

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.
Herpes Simplex Virus Type 1 and 2 Glycoprotein C Prevents Complement-Mediated Neutralization Induced by Natural Immunoglobulin M Antibody
Authors: Hook Lauren M. , Lubinski John M. , Jiang Ming , Pangburn Michael K. , Friedman Harvey M.
Published: March 2006

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) binds complement component C3b and protects virus from complement-mediated neutralization. Differences in complement interacting domains exist between gC of HSV-1 (gC1) and HSV-2 (gC2), since the amino terminus of gC1 blocks complement C5 from binding to C3b, while gC2 fails to interfere with this activity. We previously reported that neutralization of HSV-1 gC-null virus by HSV antibody-negative human serum requires activation of C5 but not of downstream components of the classical complement pathway. In this report, we evaluated whether activation of C5 is sufficient to neutralize HSV-2 gC-null virus, or whether formation of the membrane attack complex by C6 to C9 is required for neutralization. We found that activation of the classical complement pathway up to C5 was sufficient to neutralize HSV-2 gC-null virus by HSV antibody-negative human serum. We evaluated the mechanisms by which complement activation occurred in seronegative human serum. Interestingly, natural immunoglobulin M antibodies bound to virus, which triggered activation of C1q and the classical complement pathway. HSV antibody-negative sera obtained from four individuals differed over an approximately 10-fold range in their potency for complement-mediated virus neutralization. These findings indicate that humans differ in the ability of their innate immune systems to neutralize HSV-1 or HSV-2 gC-null virus and that a critical function of gC1 and gC2 is to prevent C5 activation.
Analysis of Equid Herpesvirus 1 Strain Variation Reveals a Point Mutation of the DNA Polymerase Strongly Associated with Neuropathogenic versus Nonneuropathogenic Disease Outbreaks
Authors: Nugent J. , Birch-Machin I. , Smith K. C. , Mumford J. A. , Swann Z. , Newton J. R. , Bowden R. J. , Allen G. P. , Davis-Poynter N.
Published: March 2006

Equid herpesvirus 1 (EHV-1) can cause a wide spectrum of diseases ranging from inapparent respiratory infection to the induction of abortion and, in extreme cases, neurological disease resulting in paralysis and ultimately death. It has been suggested that distinct strains of EHV-1 that differ in pathogenic capacity circulate in the field. In order to investigate this hypothesis, it was necessary to identify genetic markers that allow subgroups of related strains to be identified. We have determined all of the genetic differences between a neuropathogenic strain (Ab4) and a nonneuropathogenic strain (V592) of EHV-1 and developed PCR/sequencing procedures enabling differentiation of EHV-1 strains circulating in the field. The results indicate the occurrence of several major genetic subgroups of EHV-1 among isolates recovered from outbreaks over the course of 30 years, consistent with the proposal that distinct strains of EHV-1 circulate in the field. Moreover, there is evidence that certain strain groups are geographically restricted, being recovered predominantly from outbreaks occurring in either North America or Europe. Significantly, variation of a single amino acid of the DNA polymerase is strongly associated with neurological versus nonneurological disease outbreaks. Strikingly, this variant amino acid occurs at a highly conserved position for herpesvirus DNA polymerases, suggesting an important functional role.
Trim-Cyclophilin A Fusion Proteins Can Restrict Human Immunodeficiency Virus Type 1 Infection at Two Distinct Phases in the Viral Life Cycle
Authors: Yap Melvyn W. , Dodding Mark P. , Stoye Jonathan P.
Published: March 2006

The Trim5α protein from several primates restricts retroviruses in a capsid (CA)-dependent manner. In owl monkeys, the B30.2 domain of Trim5 has been replaced by cyclophilin A (CypA) following a retrotransposition. Restriction of human immunodeficiency virus type 1 (HIV-1) by the resulting Trim5-CypA fusion protein depends on CA binding to CypA, suggesting both that the B30.2 domain might be involved in CA binding and that the tripartite
R
ING motif,
B
-BOX, and
c
oiled
c
oil (RBCC) motif domain can function independently of the B30.2 domain in restriction. To investigate the potential of RBCCs from other Trims to participate in restricting HIV-1, CypA was fused to the RBCC of Trim1, Trim18, and Trim19 and tested for restriction. Despite low identity within the RBCC domain, all fusion proteins were found to restrict HIV-1 but not the nonbinding G89V mutant, indicating that the overall structure of RBCC and not its primary sequence was important for the restriction function. The critical interaction between CA and Trim-CypA appears to take place soon after viral entry. Quantitative PCR analysis of viral reverse transcriptase products revealed that the different fusion proteins block HIV-1 at two distinct stages of its life cycle, either prior to reverse transcription or just before integration. With Trim1 and Trim18, this timing is dependent on the length of the Trim component of the fusion protein. These observations suggest that restriction factor binding can have different mechanistic consequences.
Dissecting the Regions of Virion-Associated Kaposi's Sarcoma-Associated Herpesvirus Complement Control Protein Required for Complement Regulation and Cell Binding
Authors: Spiller O. B. , Mark L. , Blue C. E. , Proctor D. G. , Aitken J. A. , Blom A. M. , Blackbourn D. J.
Published: March 2006

Complement, which bridges innate and adaptive immune responses as well as humoral and cell-mediated immunity, is antiviral. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a lytic cycle protein called KSHV complement control protein (KCP) that inhibits activation of the complement cascade. It does so by regulating C3 convertases, accelerating their decay, and acting as a cofactor for factor I degradation of C4b and C3b, two components of the C3 and C5 convertases. These complement regulatory activities require the short consensus repeat (SCR) motifs, of which KCP has four (SCRs 1 to 4). We found that in addition to KCP being expressed on the surfaces of experimentally infected endothelial cells, it is associated with the envelope of purified KSHV virions, potentially protecting them from complement-mediated immunity. Furthermore, recombinant KCP binds heparin, an analogue of the known KSHV cell attachment receptor heparan sulfate, facilitating infection. Treating virus with an anti-KCP monoclonal antibody (MAb), BSF8, inhibited KSHV infection of cells by 35%. Epitope mapping of MAb BSF8 revealed that it binds within SCR domains 1 and 2, also the region of the protein involved in heparin binding. This MAb strongly inhibited classical C3 convertase decay acceleration by KCP and cofactor activity for C4b cleavage but not C3b cleavage. Our data suggest similar topological requirements for cell binding by KSHV, heparin binding, and regulation of C4b-containing C3 convertases but not for factor I-mediated cleavage of C3b. Importantly, they suggest KCP confers at least two functions on the virion: cell binding with concomitant infection and immune evasion.
Mucosal Immunization with Surface-Displayed Severe Acute Respiratory Syndrome Coronavirus Spike Protein onLactobacillus caseiInduces Neutralizing Antibodies in Mice
Authors: Lee Jong-Soo , Poo Haryoung , Han Dong P. , Hong Seung-Pyo , Kim Kwang , Cho Michael W. , Kim Eun , Sung Moon-Hee , Kim Chul-Joong
Published: March 2006

Induction of mucosal immunity may be important for preventing SARS-CoV infections. For safe and effective delivery of viral antigens to the mucosal immune system, we have developed a novel surface antigen display system for lactic acid bacteria using the poly-γ-glutamic acid synthetase A protein (PgsA) ofBacillus subtilisas an anchoring matrix. Recombinant fusion proteins comprised of PgsA and the Spike (S) protein segments SA (residues 2 to 114) and SB (residues 264 to 596) were stably expressed inLactobacillus casei. Surface localization of the fusion protein was verified by cellular fractionation analyses, immunofluorescence microscopy, and flow cytometry. Oral and nasal inoculations of recombinantL. caseiinto mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA, as demonstrated by enzyme-linked immunosorbent assays using S protein peptides. More importantly, these antibodies exhibited potent neutralizing activities against severe acute respiratory syndrome (SARS) pseudoviruses. Orally immunized mice mounted a greater neutralizing-antibody response than those immunized intranasally. Three new neutralizing epitopes were identified on the S protein using a peptide neutralization interference assay (residues 291 to 308, 520 to 529, and 564 to 581). These results indicate that mucosal immunization with recombinantL. caseiexpressing SARS-associated coronavirus S protein on its surface provides an effective means for eliciting protective immune response against the virus.