bluuebunny/crossref_metadata_2025 huggingface dataset:
Global Topology Analysis of the
Escherichia coli
Inner Membrane Proteome
Authors: Daley Daniel O. , Rapp Mikaela , Granseth Erik , Melén Karin , Drew David , von Heijne Gunnar
Published: May 2005

The protein complement of cellular membranes is notoriously resistant to standard proteomic analysis and structural studies. As a result, membrane proteomes remain ill-defined. Here, we report a global topology analysis of the
Escherichia coli
inner membrane proteome. Using C-terminal tagging with the alkaline phosphatase and green fluorescent protein, we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins. By constraining a topology prediction algorithm with this data, we derived high-quality topology models for the 601 proteins, providing a firm foundation for future functional studies of this and other membrane proteomes. We also estimated the overexpression potential for 397 green fluorescent protein fusions; the results suggest that a large fraction of all inner membrane proteins can be produced in sufficient quantities for biochemical and structural work.
The Importance of Threading Dislocations on the Motion of Domain Boundaries in Thin Films
Authors: Gabaly Farid El , Ling Wai Li W. , McCarty Kevin F. , de la Figuera Juan
Published: May 2005

Thin films often present domain structures whose detailed evolution is a subject of debate. We analyze the evolution of copper films, which contain both rotational and stacking domains, on ruthenium. Real-time observation by low-energy electron microscopy shows that the stacking domains evolve in a seemingly complex way. Not only do the stacking boundaries move in preferred directions, but their motion is extremely uneven and they become stuck when they reach rotational boundaries. We show that this behavior occurs because the stacking-boundary motion is impeded by threading dislocations. This study underscores how the coarse-scale evolution of thin films can be controlled by defects.
Firearm Violence Exposure and Serious Violent Behavior
Authors: Bingenheimer Jeffrey B. , Brennan Robert T. , Earls Felton J.
Published: May 2005

To estimate the cause-effect relationship between exposure to firearm violence and subsequent perpetration of serious violence, we applied the analytic method of propensity stratification to longitudinal data on adolescents residing in Chicago, Illinois. Results indicate that exposure to firearm violence approximately doubles the probability that an adolescent will perpetrate serious violence over the subsequent 2 years.
Simultaneous Inhibition and Redistribution of Spontaneous Light Emission in Photonic Crystals
Authors: Fujita Masayuki , Takahashi Shigeki , Tanaka Yoshinori , Asano Takashi , Noda Susumu
Published: May 2005

Inhibiting spontaneous light emission and redistributing the energy into useful forms are desirable objectives for advances in various fields, including photonics, illuminations, displays, solar cells, and even quantum-information systems. We demonstrate both the “inhibition” and “redistribution” of spontaneous light emission by using two-dimensional (2D) photonic crystals, in which the refractive index is changed two-dimensionally. The overall spontaneous emission rate is found to be reduced by a factor of 5 as a result of the 2D photonic bandgap effect. Simultaneously, the light energy is redistributed from the 2D plane to the direction normal to the photonic crystal.
Amalthea's Density Is Less Than That of Water
Authors: Anderson John D. , Johnson Torrence V. , Schubert Gerald , Asmar Sami , Jacobson Robert A. , Johnston Douglas , Lau Eunice L. , Lewis George , Moore William B. , Taylor Anthony , Thomas Peter C. , Weinwurm Gudrun
Published: May 2005

Radio Doppler data from the Galileo spacecraft's encounter with Amalthea, one of Jupiter's small inner moons, on 5 November 2002 yield a mass of (2.08 ± 0.15) × 10
18
kilograms. Images of Amalthea from two Voyager spacecraft in 1979 and Galileo imaging between November 1996 and June 1997 yield a volume of (2.43 ± 0.22) × 10
6
cubic kilometers. The satellite thus has a density of 857 ± 99 kilograms per cubic meter. We suggest that Amalthea is porous and composed of water ice, as well as rocky material, and thus formed in a cold region of the solar system, possibly not at its present location near Jupiter.
Direct Radiometric Dating of Hydrocarbon Deposits Using Rhenium-Osmium Isotopes
Authors: Selby David , Creaser Robert A.
Published: May 2005

Rhenium-osmium (Re-Os) data from migrated hydrocarbons establish the timing of petroleum emplacement for the giant oil sand deposits of Alberta, Canada, at 112 ± 5.3 million years ago. This date does not support models that invoke oil generation and migration for these deposits in the Late Cretaceous. Most Re-Os data from a variety of deposits within the giant hydrocarbon system show similar characteristics, supporting the notion of a single source for these hydrocarbons. The Re-Os data disqualify Cretaceous rocks as the primary hydrocarbon source but suggest an origin from older source rocks. This approach should be applicable to dating oil deposits worldwide.
Sifting Myths for Truths About Our World
Authors: Baird Abigail A.
Published: May 2005

When They Severed Earth from Sky
How the Human Mind Shapes Myth.
By Elizabeth Wayland Barber and Paul T. Barber
. Princeton University Press, Princeton, NJ, 2004. 310 pp. $29.95, £18.95. ISBN 0-691-09986-3.

The authors, archaeologists, argue that myths contain elements of truth about the natural world and that we can use them as artifacts of sorts.
One Cause for All Confabulations?
Authors: Schnider Armin
Published: May 2005

Brain Fiction
Self-Deception and the Riddle of Confabulation.
By William Hirstein
. MIT Press, Cambridge, MA, 2005. 301 pp. $35, £22.95. ISBN 0-262-08338-8. Philosophical Psychopathology: Disorders in Mind.

Exploring what causes some people to construct false answers that they genuinely believe to be true, the author argues that these considerations of these causes tell us something important about the structure of human intellect.
Structural Bioinformatics-Based Design of Selective, Irreversible Kinase Inhibitors
Authors: Cohen Michael S. , Zhang Chao , Shokat Kevan M. , Taunton Jack
Published: May 2005

The active sites of 491 human protein kinase domains are highly conserved, which makes the design of selective inhibitors a formidable challenge. We used a structural bioinformatics approach to identify two selectivity filters, a threonine and a cysteine, at defined positions in the active site of p90 ribosomal protein S6 kinase (RSK). A fluoromethylketone inhibitor, designed to exploit both selectivity filters, potently and selectively inactivated RSK1 and RSK2 in mammalian cells. Kinases with only one selectivity filter were resistant to the inhibitor, yet they became sensitized after genetic introduction of the second selectivity filter. Thus, two amino acids that distinguish RSK from other protein kinases are sufficient to confer inhibitor sensitivity.
Proteolytic Processing of Sapovirus ORF1 Polyprotein
Authors: Oka Tomoichiro , Katayama Kazuhiko , Ogawa Satoko , Hansman Grant S. , Kageyama Tsutomu , Ushijima Hiroshi , Miyamura Tatsuo , Takeda Naokazu
Published: May 2005

The genome of
Sapovirus
(SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in
1171
Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH
2
-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family
Caliciviridae
, especially to rabbit hemorrhagic disease virus, a member of the genus
Lagovirus
.
Dendritic Cell Precursors Are Permissive to Dengue Virus and Human Immunodeficiency Virus Infection
Authors: Kwan Wing-Hong , Helt Anna-Marija , Marañón Concepción , Barbaroux Jean-Baptiste , Hosmalin Anne , Harris Eva , Fridman Wolf H. , Mueller Chris G. F.
Published: May 2005

CD14+interstitial cells reside beneath the epidermis of skin and mucosal tissue and may therefore play an important role in viral infections and the shaping of an antiviral immune response. However, in contrast to dendritic cells (DC) or blood monocytes, these antigen-presenting cells (APC) have not been well studied. We have previously described long-lived CD14+cells generated from CD34+hematopoietic progenitors, which may represent model cells for interstitial CD14+APC. Here, we show that these cells carry DC-SIGN and differentiate into immature DC in the presence of granulocyte-macrophage colony-stimulating factor. We have compared the CD14+cells and the DC derived from these cells with respect to dengue virus and human immunodeficiency virus type 1 (HIV-1) infection. Both cell types are permissive to dengue virus infection, but the CD14+cells secrete the anti-inflammatory cytokine interleukin 10 and no tumor necrosis factor alpha. Regarding HIV, the CD14+cells are permissive to HIV-1, release higher p24 levels than the derived DC, and more efficiently activate HIV Pol-specific CD8+memory T cells. The CD14+DC precursors infected with either virus retain their DC differentiation potential. The results suggest that interstitial CD14+APC may contribute to HIV-1 and dengue virus infection and the shaping of an antiviral immune response.
Chimeric Dengue 2 PDK-53/West Nile NY99 Viruses Retain the Phenotypic Attenuation Markers of the Candidate PDK-53 Vaccine Virus and Protect Mice against Lethal Challenge with West Nile Virus
Authors: Huang Claire Y.-H. , Silengo Shawn J. , Whiteman Melissa C. , Kinney Richard M.
Published: May 2005

Chimeric dengue serotype 2/West Nile (D2/WN) viruses expressing prM-E of WN NY99 virus in the genetic background of wild-type D2 16681 virus and two candidate D2 PDK-53 vaccine variants (PDK53-E and PDK53-V) were engineered. The viability of the D2/WN viruses required incorporation of the WN virus-specific signal sequence for prM. Introduction of two mutations at M-58 and E-191 in the chimeric cDNA clones further improved the viability of the chimeras constructed in all three D2 carriers. Two D2/WN chimeras (D2/WN-E2 and -V2) engineered in the backbone of the PDK53-E and -V viruses retained all of the PDK-53 vaccine characteristic phenotypic markers of attenuation and were immunogenic in mice and protected mice from a high-dose 10
7
PFU challenge with wild-type WN NY99 virus. This report further supports application of the genetic background of the D2 PDK-53 virus as a carrier for development of live-attenuated, chimeric flavivirus vaccines in general and the development of a chimeric D2/WN vaccine virus against WN disease in particular.
Double-Stranded RNA Binding by Human Cytomegalovirus pTRS1
Authors: Hakki Morgan , Geballe Adam P.
Published: May 2005

The human cytomegalovirus (HCMV) TRS1 and IRS1 genes rescue replication of vaccinia virus (VV) that has a deletion of the double-stranded RNA binding protein gene E3L (VVΔE3L). Like E3L, these HCMV genes block the activation of key interferon-induced, double-stranded RNA (dsRNA)-activated antiviral pathways. We investigated the hypothesis that the products of these HCMV genes act by binding to dsRNA. pTRS1 expressed by cell-free translation or by infection of mammalian cells with HCMV or recombinant VV bound to dsRNA. Competition experiments revealed that pTRS1 preferentially bound to dsRNA compared to double-stranded DNA or single-stranded RNA. 5′- and 3′-end deletion analyses mapped the TRS1 dsRNA-binding domain to amino acids 74 through 248, a region of identity to pIRS1 that contains no homology to known dsRNA-binding proteins. Deletion of the majority of this region (Δ86-246) completely abrogated dsRNA binding. To determine the role of the dsRNA-binding domain in the rescue of VVΔE3L replication, wild-type or deletion mutants of TRS1 were transfected into HeLa cells, which were then infected with VVΔE3L. While full-length TRS1 rescued VVΔE3L replication, deletion mutants affecting a carboxy-terminal region of TRS1 that is not required for dsRNA binding failed to rescue VVΔE3L. Analyses of stable cell lines revealed that the carboxy-terminal domain is necessary to prevent the shutoff of protein synthesis and the phosphorylation of eIF2α after VVΔE3L infection. Thus, pTRS1 contains an unconventional dsRNA-binding domain at its amino terminus, but a second function involving the carboxy terminus is also required for countering host cell antiviral responses.
BZLF1 Activation of the Methylated Form of the
BRLF1
Immediate-Early Promoter Is Regulated by BZLF1 Residue 186
Authors: Bhende Prasanna M. , Seaman William T. , Delecluse Henri-Jacques , Kenney Shannon C.
Published: May 2005

The Epstein-Barr virus (EBV) genome is highly methylated in latently infected cells. We recently reported that the EBV immediate-early (IE) protein BZLF1 (Z) preferentially binds to and activates transcription from the methylated form of the
BRLF1
IE gene promoter (Rp). We now report that serine residue 186 in the Z DNA-binding domain plays an important role in the ability of Z to bind to and activate methylated Rp. A Z mutant containing an alanine residue at position 186 [Z(S186A)] was significantly defective in binding to methylated, as well as unmethylated, ZREs (Z-responsive elements) in Rp and was unable to activate lytic EBV gene transcription from the methylated or demethylated form of the viral genome. A Z mutant containing threonine at residue 186 [Z(S186T)] bound only to the methylated form of the ZRE-2 site in Rp and induced lytic EBV gene transcription from the methylated, but not demethylated, form of the viral genome. The defect in both of these mutants was primarily due to an inability to activate the Rp in the context of the viral genome. Finally, a Z mutant containing an aspartic acid at position 186 [Z(S186D)] did not bind to either the consensus AP-1 site or to the methylated or unmethylated Rp ZRE-2 site and did not induce lytic gene transcription. These results indicate that replacement of serine with threonine at residue 186 in the Z DNA-binding domain differentially affects its ability to reactivate the unmethylated, versus methylated, viral genome.
Suppression of Virus Load by Highly Active Antiretroviral Therapy in Rhesus Macaques Infected with a Recombinant Simian Immunodeficiency Virus Containing Reverse Transcriptase from Human Immunodeficiency Virus Type 1
Authors: North Thomas W. , Van Rompay Koen K. A. , Higgins Joanne , Matthews Timothy B. , Wadford Debra A. , Pedersen Niels C. , Schinazi Raymond F.
Published: May 2005

We have modeled highly active antiretroviral therapy (HAART) for AIDS in rhesus macaques infected with a chimera (RT-SHIV) of simian immunodeficiency virus containing reverse transcriptase from human immunodeficiency virus type-1 (HIV-1). Seven RT-SHIV-infected macaques were treated with a combination of efavirenz (200 mg orally once daily), lamivudine (8 mg/kg subcutaneously once daily), and tenofovir (30 mg/kg subcutaneously once daily). Plasma viral RNA levels in all animals were reduced by more than 1,000-fold after 4 weeks and, in six of the seven animals, were reduced to undetectable levels after 10 weeks. Virus loads increased slightly between 12 and 16 weeks of treatment, associated with problems with the administration of efavirenz. After a change in the method of efavirenz administration, virus loads declined again and remained undetectable in the majority of animals for the duration of therapy. Treatment was stopped for three animals after 36 weeks of therapy, and virus loads increased rapidly. Posttreatment RT-SHIV isolates had no mutations associated with resistance to any of the three drugs. Efavirenz treatment was stopped, but lamivudine and tenofovir treatment for two other macaques was continued. The virus load in one of these two animals rebounded; virus from this animal was initially free of drug-resistance mutations but acquired the K65R mutation in reverse transcriptase at 11 weeks after efavirenz treatment was withdrawn. These results mimic HAART of HIV-1-infected humans. The RT-SHIV/rhesus macaque model should be useful for studies of tissue reservoirs and sites of residual replication that are not possible or practical with humans.
Epstein-Barr Virus LMP2A Alters In Vivo and In Vitro Models of B-Cell Anergy, but Not Deletion, in Response to Autoantigen
Authors: Swanson-Mungerson Michelle A. , Caldwell Robert G. , Bultema Rebecca , Longnecker Richard
Published: May 2005

A significant percentage of the population latently harbors Epstein-Barr virus (EBV) in B cells. One EBV-encoded protein, latent membrane protein 2A (LMP2A), is expressed in tissue culture models of EBV latent infection, in human infections, and in many of the EBV-associated proliferative disorders. LMP2A constitutively activates proteins involved in the B-cell receptor (BCR) signal transduction cascade and inhibits the antigen-induced activation of these proteins. In the present study, we investigated whether LMP2A alters B-cell receptor signaling in primary B cells in vivo and in vitro. LMP2A does not inhibit antigen-induced tolerance in response to strong stimuli in an in vivo tolerance model in which B cells are reactive to self-antigen. In contrast, LMP2A bypasses anergy induction in response to low levels of soluble hen egg lysozyme (HEL) both in vivo and in vitro as determined by the ability of LMP2A-expressing HEL-specific B cells to proliferate and induce NF-κB nuclear translocation after exposure to low levels of antigen. Furthermore, LMP2A induces NF-κB nuclear translocation independent of BCR cross-linking. Since NF-κB is required to bypass tolerance induction, this LMP2A-dependent NF-κB activation may complete the tolerogenic signal induced by low levels of soluble HEL. Overall, the findings suggest that LMP2A may not inhibit BCR-induced signals under all conditions as previously suggested by studies with EBV immortalized B cells.
Vaccinia Virus Induces Strong Immunoregulatory Cytokine Production in Healthy Human Epidermal Keratinocytes: a Novel Strategy for Immune Evasion
Authors: Liu Luzheng , Xu Zhan , Fuhlbrigge Robert C. , Peña-Cruz Victor , Lieberman Judy , Kupper Thomas S.
Published: May 2005

Iatrogenic cutaneous infection with vaccinia virus (VV) and naturally occurring systemic infection with variola virus both lead to the characteristic skin “pox” lesions. Despite significant medical experience with both viruses, surprisingly little is understood about the interactions between these poxviruses and healthy resident skin cells. In recent years, it has become clear that skin plays an essential role in modulating both innate and adaptive immune responses, in part by producing and responding to a variety of cytokines and chemokines upon stimulation. Antagonists of many of these compounds are encoded in poxvirus genomes. Infection of skin cells with poxvirus may lead to a unique pattern of cytokine and chemokine production that might alter the cutaneous immune surveillance function. In this study, we infected primary cultures of human skin cells with VV and monitored antigen expression, virus replication, and cytokine production from the infected cells. While T cells, Langerhans cells, and dermal dendritic cells were infected abortively, keratinocytes, dermal fibroblasts, and dermal microvascular endothelial cells (HMVEC-d) all supported the complete virus life cycle. In contrast to the robust viral replication in fibroblasts and HMVEC-d, only limited viral replication was observed in keratinocytes. Importantly, VV infection of keratinocytes led to up-regulation of immunoregulatory and Th2 cytokines, including transforming growth factor β, interleukin-10 (IL-10), and IL-13. We propose that the rapid induction of keratinocyte Th2 and immunoregulatory cytokines represents a poxvirus strategy to evade immune surveillance, and the limited viral multiplication in keratinocytes may be a protective mechanism to help the immune system “win the race.”
A Virus-Encoded Inhibitor That Blocks RNA Interference in Mammalian Cells
Authors: Sullivan Christopher S. , Ganem Don
Published: May 2005

Nodamura virus (NoV) is a small RNA virus that is infectious for insect and mammalian hosts. We have developed a highly sensitive assay of RNA interference (RNAi) in mammalian cells that shows that the NoV B2 protein functions as an inhibitor of RNAi triggered by either short hairpin RNAs or small interfering RNAs. In the cell, NoV B2 binds to pre-Dicer substrate RNA and RNA-induced silencing complex (RISC)-processed RNAs and inhibits the Dicer cleavage reaction and, potentially, one or more post-Dicer activities. In vitro, NoV B2 inhibits Dicer-mediated RNA cleavage in the absence of any other host factors and specifically binds double-stranded RNAs corresponding in structure to Dicer substrates and products. Its abilities to bind to Dicer precursor and post-Dicer RISC-processed RNAs suggest a mechanism of inhibition that is unique among known viral inhibitors of RNAi.
Universal Influenza B Vaccine Based on the Maturational Cleavage Site of the Hemagglutinin Precursor
Authors: Bianchi Elisabetta , Liang Xiaoping , Ingallinella Paolo , Finotto Marco , Chastain Michael A. , Fan Jiang , Fu Tong-Ming , Song Hong Chang , Horton Melanie S. , Freed Daniel C. , Manger Walter , Wen Emily , Shi Li , Ionescu Roxana , Price Colleen , Wenger Marc , Emini Emilio A. , Cortese Riccardo , Ciliberto Gennaro , Shiver John W. , Pessi Antonello
Published: May 2005

Conventional influenza vaccines can prevent infection, but their efficacy depends on the degree of antigenic “match” between the strains used for vaccine preparation and those circulating in the population. A universal influenza vaccine based on invariant regions of the virus, able to provide broadly cross-reactive protection, without requiring continuous manufacturing update, would solve a major medical need. Since the temporal and geographical dominance of the influenza virus type and/or subtype (A/H3, A/H1, or B) cannot yet be predicted, a universal vaccine, like the vaccines currently in use, should include both type A and type B influenza virus components. However, while encouraging preclinical data are available for influenza A virus, no candidate universal vaccine is available for influenza B virus. We show here that a peptide conjugate vaccine, based on the highly conserved maturational cleavage site of the HA0precursor of the influenza B virus hemagglutinin, can elicit a protective immune response against lethal challenge with viruses belonging to either one of the representative, non-antigenically cross-reactive influenza B virus lineages. We demonstrate that protection by the HA0vaccine is mediated by antibodies, probably through effector mechanisms, and that a major part of the protective response targets the most conserved region of HA0, the P1 residue of the scissile bond and the fusion peptide domain. In addition, we present preliminary evidence that the approach can be extended to influenza A virus, although the equivalent HA0conjugate is not as efficacious as for influenza B virus.
The Minor Receptor Group of Human Rhinovirus (HRV) Includes HRV23 and HRV25, but the Presence of a Lysine in the VP1 HI Loop Is Not Sufficient for Receptor Binding
Authors: Vlasak Marketa , Roivainen Merja , Reithmayer Manuela , Goesler Irene , Laine Pia , Snyers Luc , Hovi Tapani , Blaas Dieter
Published: May 2005

Like all 10 minor receptor group human rhinoviruses (HRVs), HRV23 and HRV25, previously classified as major group viruses, are neutralized by maltose binding protein (MBP)-V33333 (a soluble recombinant concatemer of five copies of repeat 3 of the very-low-density lipoprotein receptor fused to MBP), bind to low-density lipoprotein receptor in virus overlay blots, and replicate in intercellular adhesion molecule 1 (ICAM-1)-negative COS-7 cells. From phylogenetic analysis of capsid protein VP1-coding sequences, they are also known to cluster together with other minor group strains. Therefore, they belong to the minor group; there are now 12 minor group and 87 major group HRV serotypes. Sequence comparison of the VP1 capsid proteins of all HRVs revealed that the lysine in the HI loop, strictly conserved in the 12 minor group HRVs, is also present in 9 major group serotypes that are neutralized by soluble ICAM-1. Despite the presence of this lysine, they are not neutralized by MBP-V33333 and fail to replicate in COS-7 cells and in HeLa cells in the presence of an ICAM-1-blocking antibody. These nine serotypes are therefore “true” major group viruses.
The Exonuclease and Host Shutoff Functions of the SOX Protein of Kaposi's Sarcoma-Associated Herpesvirus Are Genetically Separable
Authors: Glaunsinger Britt , Chavez Leonard , Ganem Don
Published: May 2005

The Kaposi's sarcoma-associated herpesvirus (KSHV) SOX protein, encoded by ORF37, promotes shutoff of host cell gene expression during lytic viral replication by dramatically impairing mRNA accumulation. SOX is the KSHV homolog of the alkaline exonuclease of other herpesviruses, which has been shown to function as a DNase involved in processing and packaging the viral genome. Although the exonuclease activity of these proteins is widely conserved across all herpesviruses, the host shutoff activity observed for KSHV SOX is not. We show here that SOX expression sharply reduces the half-life of target mRNAs. Extensive mutational analysis reveals that the DNase and host shutoff activities of SOX are genetically separable. Lesions affecting the DNase activity cluster in conserved regions of the protein, but residues critical for mRNA degradation are not conserved across the viral family. Additionally, we present evidence suggesting that the two different functions of SOX occur within distinct cellular compartments—DNase activity in the nucleus and host shutoff activity in the cytoplasm.
Identification of Genogroup I and Genogroup II Broadly Reactive Epitopes on the Norovirus Capsid
Authors: Parker Tracy Dewese , Kitamoto Noritoshi , Tanaka Tomoyuki , Hutson Anne M. , Estes Mary K.
Published: May 2005

Norwalk virus, a member of the familyCaliciviridae, is an important cause of acute epidemic nonbacterial gastroenteritis. Norwalk and related viruses are classified in a separate genus ofCaliciviridaecalledNorovirus, which is comprised of at least three genogroups based on sequence differences. Many of the currently available immunologic reagents used to study these viruses are type specific, which limits the identification of antigenically distinct viruses in detection assays. Identification of type-specific and cross-reactive epitopes is essential for designing broadly cross-reactive diagnostic assays and dissecting the immune response to calicivirus infection. To address this, we have mapped the epitopes on the norovirus capsid protein for both a genogroup I-cross-reactive monoclonal antibody and a genogroup II-cross-reactive monoclonal antibody by use of norovirus deletion and point mutants. The epitopes for both monoclonal antibodies mapped to the C-terminal P1 subdomain of the capsid protein. Although the genogroup I-cross-reactive monoclonal antibody was previously believed to recognize a linear epitope, our results indicate that a conformational component of the epitope explains the monoclonal antibody's genogroup specificity. Identification of the epitopes for these monoclonal antibodies is of significance, as they are components in a commercially available norovirus-diagnostic enzyme-linked immunosorbent assay.
Adenosine Kinase Inhibition and Suppression of RNA Silencing by Geminivirus AL2 and L2 Proteins
Authors: Wang Hui , Buckley Kenneth J. , Yang Xiaojuan , Buchmann R. Cody , Bisaro David M.
Published: May 2005

Most plant viruses are initiators and targets of RNA silencing and encode proteins that suppress this adaptive host defense. The DNA-containing geminiviruses are no exception, and the AL2 protein (also known as AC2, C2, and transcriptional activator protein) encoded by members of the genus
Begomovirus
has been shown to act as a silencing suppressor. Here, a three-component,
Agrobacterium
-mediated transient assay is used to further examine the silencing suppression activity of AL2 from
Tomato golden mosaic virus
(TGMV, a begomovirus) and to determine if the related L2 protein of
Beet curly top virus
(BCTV, genus
Curtovirus
) also has suppression activity. We show that TGMV AL2, AL2
1-100
(lacking the transcriptional activation domain), and BCTV L2 can all suppress RNA silencing directed against a green fluorescent protein (GFP) reporter gene when silencing is induced by a construct expressing an inverted repeat GFP RNA (dsGFP). We previously found that these viral proteins interact with and inactivate adenosine kinase (ADK), a cellular enzyme important for adenosine salvage and methyl cycle maintenance. Using the GFP-dsGFP system, we demonstrate here that codelivery of a construct expressing an inverted repeat ADK RNA (dsADK), or addition of an ADK inhibitor (the adenosine analogue A-134974), suppresses GFP-directed silencing in a manner similar to the geminivirus proteins. In addition, AL2/L2 suppression phenotypes and nucleic acid binding properties are shown to be different from those of the RNA virus suppressors HC-Pro and p19. These findings provide strong evidence that ADK activity is required to support RNA silencing, and indicate that the geminivirus proteins suppress silencing by a novel mechanism that involves ADK inhibition. Further, since AL2
1-100
is as effective a suppressor as the full-length AL2 protein, activation and silencing suppression appear to be independent activities.
The C Terminus of the B5 Receptor for Herpes Simplex Virus Contains a Functional Region Important for Infection
Authors: Perez-Romero Pilar , Fuller A. Oveta
Published: May 2005

The expression of a previously uncharacterized human
hfl-B5
cDNA confers susceptibility for herpes simplex virus (HSV) to porcine cells and fulfills criteria as an HSV entry receptor (A. Perez, Q.-X. Li, P. Perez-Romero, G. DeLassus, S. R. Lopez, S. Sutter, N. McLaren, and A. Oveta Fuller, J. Virol. 79:7419-7430, 2005). Heptad repeats found in the B5 C terminus are predicted to form an α-helix for coiled coil structure. We used mutagenesis and synthetic peptides with wild-type and mutant sequences to examine the function of the heptad repeat motif in HSV binding and entry into porcine cells that express B5 and for infection of naturally susceptible human HEp-2 cells. B5 with point mutations predicted to disrupt the putative C-terminal coiled coil failed to mediate HSV binding and entry into porcine cells. Synthetic peptides that contain the single amino acid changes lose the blocking activity of HSV entry. We concluded that the C terminus of B5 contains a functional region that is important for the B5 receptor to mediate events in HSV entry. Structural evidence that this functional region forms coiled coil structures is under investigation. Blocking of HSV interaction with the C-terminal region of the B5 receptor is a new potential target site to intervene in the virus infection of human cells.
Localization of Regions in CD46 That Interact with Adenovirus
Authors: Gaggar Anuj , Shayakhmetov Dmitry M. , Liszewski M. Kathryn , Atkinson John P. , Lieber André
Published: May 2005

A variety of pathogens use CD46, a ubiquitously expressed membrane protein that regulates complement activation, as a cellular attachment receptor. While the CD46 binding sites of several pathogens, including measles virus,Neisseria gonorrhea, and human herpesvirus 6, have been described, the region of CD46 responsible for adenovirus binding has not been determined. In this study, we used competition experiments with known CD46 ligands, CD46-specific antibodies, and a set of CD46 mutants to localize the binding domain for the group B adenovirus serotype 35 (Ad35). Our results show that Ad35 competes with measles virus for binding to CD46 but not with complement protein C3b. We further show that this interaction is a protein-protein interaction and that N glycosylations do not critically contribute to infection with Ad35 fiber-containing Ad vectors. Our data demonstrate that the native conformation of the CCP2 domain is crucial for Ad35 binding and that the substitution of amino acids at positions 130 to 135 or 152 to 156 completely abolishes the receptor function of CD46. These regions localize to the same planar face of CD46 and likely form an extended adenovirus binding surface, since no single amino acid substitution within these areas eliminates virus binding. Finally, we demonstrate that the infection with a virus possessing human group B serotype Ad11 fibers is also mediated by the CCP2 domain. This information is important to better characterize the mechanisms of the receptor recognition by adenovirus relative to other pathogens that interact with CD46, and it may help in the design of antiviral therapeutics against adenovirus serotypes that use CD46 as a primary cellular attachment receptor.
Invasion of the Central Nervous System in a Porcine Host by Nipah Virus
Authors: Weingartl Hana , Czub Stefanie , Copps John , Berhane Yohannes , Middleton Deborah , Marszal Peter , Gren Jason , Smith Greg , Ganske Shelley , Manning Lisa , Czub Markus
Published: May 2005

Nipah virus, a newly emerged zoonotic paramyxovirus, infects a number of species. Human infections were linked to direct contact with pigs, specifically with their body fluids. Clinical signs in human cases indicated primarily involvement of the central nervous system, while in pigs the respiratory system was considered the primary virus target, with only rare involvement of the central nervous system. Eleven 5-week-old piglets were infected intranasally, orally, and ocularly with 2.5 × 10
5
PFU of Nipah virus per animal and euthanized between 3 and 8 days postinoculation. Nipah virus caused neurological signs in two out of eleven inoculated pigs. The rest of the pigs remained clinically healthy. Virus was detected in the respiratory system (turbinates, nasopharynx, trachea, bronchus, and lung in titers up to 10
5.3
PFU/g) and in the lymphoreticular system (endothelial cells of blood and lymphatic vessels, submandibular and bronchiolar lymph nodes, tonsil, and spleen with titers up to 10
6
PFU/g). Virus presence was confirmed in the nervous system of both sick and apparently healthy animals (cranial nerves, trigeminal ganglion, brain, and cerebrospinal fluid, with titers up to 10
7.7
PFU/g of tissue). Nipah virus distribution was confirmed by immunohistochemistry. The study presents novel findings indicating that Nipah virus invaded the central nervous system of the porcine host via cranial nerves as well as by crossing the blood-brain barrier after initial virus replication in the upper respiratory tract.
Mutations in the NS1 Protein of Swine Influenza Virus Impair Anti-Interferon Activity and Confer Attenuation in Pigs
Authors: Solórzano Alicia , Webby Richard J. , Lager Kelly M. , Janke Bruce H. , García-Sastre Adolfo , Richt Jürgen A.
Published: May 2005

It has been shown previously that the nonstructural protein NS1 of influenza virus is an alpha/beta interferon (IFN-α/β) antagonist, both in vitro and in experimental animal model systems. However, evidence of this function in a natural host has not yet been obtained. Here we investigated the role of the NS1 protein in the virulence of a swine influenza virus (SIV) isolate in pigs by using reverse genetics. The virulent wild-type A/Swine/Texas/4199-2/98 (TX/98) virus and various mutants encoding carboxy-truncated NS1 proteins were rescued. Growth properties of TX/98 viruses with mutated NS1, induction of IFN in tissue culture, and virulence-attenuation in pigs were analyzed and compared to those of the recombinant wild-type TX/98 virus. Our results indicate that deletions in the NS1 protein decrease the ability of the TX/98 virus to prevent IFN-α/β synthesis in pig cells. Moreover, all NS1 mutant viruses were attenuated in pigs, and this correlated with the amount of IFN-α/β induced in vitro. These data suggest that the NS1 protein of SIV is a virulence factor. Due to their attenuation, NS1-mutated swine influenza viruses might have a great potential as live attenuated vaccine candidates against SIV infections of pigs.
Abortive versus Productive Viral Infection of Dendritic Cells with a Paramyxovirus Results in Differential Upregulation of Select Costimulatory Molecules
Authors: Pejawar Sharmila S. , Parks Griffith D. , Alexander-Miller Martha A.
Published: May 2005

Dendritic cells are the most potent antigen-presenting cell for priming naive T cells. Optimal activation of T cells requires that dendritic cells undergo a process of maturation resulting in the increased expression of costimulatory molecules, such as CD40, CD86, and CD80, and the production of cytokines. In this study we analyzed the effect of infection of dendritic cells obtained from two strains of mice, BALB/c and C57BL/6, with the paramyxovirus simian virus 5 (SV5). Our results show that C57BL/6 bone marrow-derived dendritic cells (BMDC) are much more permissive to infection with SV5 at a multiplicity of infection (MOI) of 10 PFU/cell compared to BALB/c BMDC, as determined by the production of viral proteins and progeny. However, infection of BALB/c BMDC with a higher MOI of 50 PFU/cell resulted in a productive infection with the production of significant amounts of viral proteins and progeny. Regardless of the permissivity to infection, both BALB/c and C57BL/6 BMDC efficiently upregulated CD40 and CD86. However, CD80 upregulation correlated with the level of expression of viral proteins and the production of viral progeny. While secreted alpha/beta interferon was required for increased expression of all three molecules, optimal CD80 expression was dependent on an additional signal provided by a productive viral infection. These findings provide evidence that the signals controlling the expression of costimulatory molecules following viral infection are distinct. Further, they suggest that the amount of virus encountered and/or the permissivity of a dendritic cell to infection can alter the resulting maturation phenotype and functional capacity of the infected dendritic cell.
Predicting Coding Potential from Genome Sequence: Application to Betaherpesviruses Infecting Rats and Mice
Authors: Brocchieri Luciano , Kledal Thomas N. , Karlin Samuel , Mocarski Edward S.
Published: May 2005

Prediction of protein-coding regions and other features of primary DNA sequence have greatly contributed to experimental biology. Significant challenges remain in genome annotation methods, including the identification of small or overlapping genes and the assessment of mRNA splicing or unconventional translation signals in expression. We have employed a combined analysis of compositional biases and conservation together with frame-specific G+C representation to reevaluate and annotate the genome sequences of mouse and rat cytomegaloviruses. Our analysis predicts that there are at least 34 protein-coding regions in these genomes that were not apparent in earlier annotation efforts. These include 17 single-exon genes, three new exons of previously identified genes, a newly identified four-exon gene for a lectin-like protein (in rat cytomegalovirus), and 10 probable frameshift extensions of previously annotated genes. This expanded set of candidate genes provides an additional basis for investigation in cytomegalovirus biology and pathogenesis.
Herpes Simplex Virus Type 1 Enters Human Epidermal Keratinocytes, but Not Neurons, via a pH-Dependent Endocytic Pathway
Authors: Nicola Anthony V. , Hou Jean , Major Eugene O. , Straus Stephen E.
Published: May 2005

Herpes simplex virus (HSV) enters some laboratory cell lines via a pH-dependent, endocytic mechanism. We investigated whether this entry pathway is used in human cell types relevant to pathogenesis. Three different classes of lysosomotropic agents, which raise endosomal pH, blocked HSV entry into primary and transformed human keratinocytes, but not into human neurons or neuroblastoma lines. In keratinocytes, incoming HSV particles colocalized with markers of endocytic uptake. Treatment with the isoflavone genistein, an inhibitor of protein tyrosine kinases, reduced the delivery of incoming viral particles to the nuclear periphery and virus-induced gene expression in keratinocytes but not neurons. Moreover, in keratinocyte monolayer islets, HSV infected both the inner and outer cells in a genistein-sensitive manner, suggesting viral endocytosis from both basolateral and apical plasma membrane surfaces. Together, the results indicate that HSV enters human epidermal keratinocytes, but not neurons, by a low-pH, endocytic pathway that is dependent on host tyrosine phosphorylation. Thus, HSV utilizes fundamentally different cellular entry pathways to infect important target cell populations.
Iκβ-Related
vankyrin
Genes in the
Campoletis sonorensis
Ichnovirus: Temporal and Tissue-Specific Patterns of Expression in Parasitized
Heliothis virescens
Lepidopteran Hosts
Authors: Kroemer Jeremy A. , Webb Bruce A.
Published: May 2005

Polydnaviruses (PDVs) are unusual insect viruses that occur in obligate symbiotic associations with parasitic ichneumonid (ichnoviruses, or IVs) and braconid (bracoviruses, or BVs) wasps. PDVs are injected with eggs, ovarian proteins, and venom during parasitization. Following infection of cells in host tissues, viral genes are expressed and their products function to alter lepidopteran host physiology, enabling endoparasitoid development. Here we describe the
Campoletis sonorensis
IV
viral ankyrin
(
vankyrin
) gene family and its transcription. The seven members of this gene family possess ankyrin repeat domains that resemble the inhibitory domains of the
Drosophila melanogaster
NF-κβ transcription factor inhibitor (Iκβ) cactus.
vankyrin
gene expression is detected within 2 to 4 h postparasitization (p.p.) in
Heliothis virescens
hosts and reaches peak levels by 3 days p.p. Our data indicate that
vankyrin
genes from the
C. sonorensis
IV genome are differentially expressed in the tissues of parasitized hosts and can be divided into two subclasses: those that target the host fat body and those that target host hemocytes. Polyclonal antibodies raised against a fat-body targeting vankyrin detected a 19-kDa protein in crude extracts prepared from the 3 days p.p. fat body. Vankyrin-specific Abs localized to 3-day p.p. fat-body and hemocyte nuclei, suggesting a role for vankyrin proteins in the nuclei of
C. sonorensis
IV-infected cells. These data are evidence for divergent tissue specificities and targeting of multigene families in IVs. We hypothesize that PDV
vankyrin
genes may suppress NF-κβ activity during immune responses and developmental cascades in parasitized lepidopteran hosts of
C. sonorensis
.
Murine Coronavirus Evolution In Vivo: Functional Compensation of a Detrimental Amino Acid Substitution in the Receptor Binding Domain of the Spike Glycoprotein
Authors: Navas-Martin Sonia , Hingley Susan T. , Weiss Susan R.
Published: May 2005

Murine coronavirus A59 strain causes mild to moderate hepatitis in mice. We have previously shown that mutants of A59, unable to induce hepatitis, may be selected by persistent infection of primary glial cells in vitro. These in vitro isolated mutants encoded two amino acids substitutions in the spike (S) gene: Q159L lies in the putative receptor binding domain of S, and H716D, within the cleavage signal of S. Here, we show that hepatotropic revertant variants may be selected from these in vitro isolated mutants (Q159L-H716D) by multiple passages in the mouse liver. One of these mutants, hr2, was chosen for more in-depth study based on a more hepatovirulent phenotype. The S gene of hr2 (Q159L-R654H-H716D-E1035D) differed from the in vitro isolates (Q159L-H716D) in only 2 amino acids (R654H and E1035D). Using targeted RNA recombination, we have constructed isogenic recombinant MHV-A59 viruses differing only in these specific amino acids in S (Q159L-R654H-H716D-E1035D). We demonstrate that specific amino acid substitutions within the spike gene of the hr2 isolate determine the ability of the virus to cause lethal hepatitis and replicate to significantly higher titers in the liver compared to wild-type A59. Our results provide compelling evidence of the ability of coronaviruses to rapidly evolve in vivo to highly virulent phenotypes by functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein.
Analysis and Function of Prototype Foamy Virus Envelope N Glycosylation
Authors: Lüftenegger Daniel , Picard-Maureau Marcus , Stanke Nicole , Rethwilm Axel , Lindemann Dirk
Published: May 2005

The prototype foamy virus (PFV) glycoprotein, which is essential for PFV particle release, displays a highly unusual biosynthesis, resulting in posttranslational cleavage of the precursor protein into three particle-associated subunits, i.e., leader peptide (LP), surface (SU), and transmembrane (TM). Glycosidase digestion of metabolically labeled PFV particles revealed the presence of N-linked carbohydrates on all subunits. The differential sensitivity to specific glycosidases indicated that all oligosaccharides on LP and TM are of the high-mannose or hybrid type, whereas most of those attached to SU, which contribute to about 50% of its molecular weight, are of the complex type. Individual inactivation of all 15 potential N-glycosylation sites in PFV Env demonstrated that 14 are used, i.e., 1 out of 2 in LP, 10 in SU, and 3 in TM. Analysis of the individual altered glycoproteins revealed defects in intracellular processing, support of particle release, and infectivity for three mutants, having the evolutionarily conserved glycosylation sites N8 in SU or N13 and N15 in the cysteine-rich central “sheets-and-loops” region of TM inactivated. Examination of alternative mutants with mutations affecting glycosylation or surrounding sequences at these sites indicated that inhibition of glycosylation at N8 and N13 most likely is responsible for the observed replication defects, whereas for N15 surrounding sequences seem to contribute to a temperature-sensitive phenotype. Taken together these data demonstrate that PFV Env and in particular the SU subunit are heavily N glycosylated and suggest that although most carbohydrates are dispensable individually, some evolutionarily conserved sites are important for normal Env function of FV isolates from different species.