bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multisite Study of Cryptosporidiosis in Children with Diarrhea in India
Authors: Ajjampur Sitara Swarna Rao , Liakath Farzana Begum , Kannan Arun , Rajendran Priya , Sarkar Rajiv , Moses Prabhakar Devarajan , Simon Anna , Agarwal Indira , Mathew Ann , O'Connor Roberta , Ward Honorine , Kang Gagandeep 
Published: April 2010

ABSTRACT

Cryptosporidium
spp., a common cause of diarrhea in children, were investigated in the first multisite study in India. Diarrheal stools from hospitalized children aged <5 years from Delhi, Trichy, and Vellore were analyzed by microscopy, PCR-restriction fragment length polymorphism (RFLP), and/or sequencing at the small-subunit (SSU) rRNA and
Cpgp40/15
loci for species determination and subgenotyping, respectively. Seventy of 2,579 (2.7%) children, 75% of whom were <2 years old, had cryptosporidial diarrhea as determined by microscopy. Genotyping and subgenotyping showed that
C
ryptosporidium
hominis
was the most commonly identified species (59/67 children), and subgenotypes Ie, Ia, Ib, and Id were common in all centers. A novel
C. parvum
subgenotype, IIn, was identified in Vellore. Meteorological analysis revealed a higher rate of cryptosporidial positivity during hotter and drier weather in Delhi.
Selection of a Rare Neutralization-Resistant Variant following Passive Transfer of Convalescent Immune Plasma in Equine Infectious Anemia Virus-Challenged SCID Horses
Authors: Taylor Sandra D. , Leib Steven R. , Carpenter Susan , Mealey Robert H. 
Published: April 2010

ABSTRACTVaccines preventing HIV-1 infection will likely elicit antibodies that neutralize diverse strains. However, the capacity for lentiviruses to escape broadly neutralizing antibodies (NAbs) is not completely understood, nor is it known whether NAbs alone can control heterologous infection. Here, we determined that convalescent immune plasma from a horse persistently infected with equine infectious anemia virus (EIAV) neutralized homologous virus and several envelope variants containing heterologous principal neutralizing domains (PND). Plasma was infused into young horses (foals) affected with severe combined immunodeficiency (SCID), followed by challenge with a homologous EIAV stock. Treated SCID foals were protected against clinical disease, with complete prevention of infection occurring in one foal. In three SCID foals, a novel neutralization-resistant variant arose that was found to preexist at a low frequency in the challenge inoculum. In contrast, SCID foals infused with nonimmune plasma developed acute disease associated with high levels of the predominant challenge virus. Following transfer to an immunocompetent horse, the neutralization-resistant variant induced a single febrile episode and was subsequently controlled in the absence of type-specific NAb. Long-term control was associated with the presence of cytotoxic T lymphocytes (CTL). Our results demonstrate that immune plasma with neutralizing activity against heterologous PND variants can prevent lentivirus infection and clinical disease in the complete absence of T cells. Importantly, however, rare neutralization-resistant envelope variants can replicatein vivounder relatively broad selection pressure, highlighting the need for protective lentivirus vaccines to elicit NAb responses with increased breadth and potency and/or CTL that target conserved epitopes.
An LYPSL Late Domain in the Gag Protein Contributes to the Efficient Release and Replication of Rous Sarcoma Virus
Authors: Dilley Kari A. , Gregory Devon , Johnson Marc C. , Vogt Volker M. 
Published: April 2010

ABSTRACT

The efficient release of newly assembled retrovirus particles from the plasma membrane requires the recruitment of a network of cellular proteins (ESCRT machinery) normally involved in the biogenesis of multivesicular bodies and in cytokinesis. Retroviruses and other enveloped viruses recruit the ESCRT machinery through three classes of short amino acid consensus sequences termed late domains: PT/SAP, PPXY, and LYPX
n
L. The major late domain of Rous sarcoma virus (RSV) has been mapped to a PPPY motif in Gag that binds members of the Nedd4 family of ubiquitin ligases. RSV Gag also contains a second putative late domain motif, LYPSL, positioned 5 amino acids downstream of PPPY. LYPX
n
L motifs have been shown to support budding in other retroviruses by binding the ESCRT adaptor protein Alix. To investigate a possible role of the LYPSL motif in RSV budding, we constructed PPPY and LYPSL mutants in the context of an infectious virus and then analyzed the budding rates, spreading profiles, and budding morphology. The data imply that the LYPSL motif acts as a secondary late domain and that its role in budding is amplified in the absence of a fully functional PPPY motif. The LYPXL motif proved to be a stronger late domain when an aspartic acid was substituted for the native serine, recapitulating the properties of the LYPDL late domain of equine infectious anemia virus. The overexpression of human Alix in the absence of a fully functional PPPY late domain partially rescued both the viral budding rate and viral replication, supporting a model in which the RSV LYPSL motif mediates budding through an interaction with the ESCRT adaptor protein Alix.
The RING Finger Domain of Varicella-Zoster Virus ORF61p Has E3 Ubiquitin Ligase Activity That Is Essential for Efficient Autoubiquitination and Dispersion of Sp100-Containing Nuclear Bodies
Authors: Walters Matthew S. , Kyratsous Christos A. , Silverstein Saul J. 
Published: April 2010

ABSTRACT
Varicella zoster virus encodes an immediate-early (IE) protein termed ORF61p that is orthologous to the herpes simplex virus IE protein ICP0. Although these proteins share several functional properties, ORF61p does not fully substitute for ICP0. The greatest region of similarity between these proteins is a RING finger domain. We demonstrate that disruption of the ORF61p RING finger domain by amino acid substitution (Cys19Gly) alters ORF61p intranuclear distribution and abolishes ORF61p-mediated dispersion of Sp100-containing nuclear bodies. In addition, we demonstrate that an intact ORF61p RING finger domain is necessary for E3 ubiquitin ligase activity and is required for autoubiquitination and regulation of protein stability.
Live-Cell Coimaging of the Genomic RNAs and Gag Proteins of Two Lentiviruses
Authors: Kemler Iris , Meehan Anne , Poeschla Eric M. 
Published: April 2010

ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) Gag and genomic RNA determinants required for encapsidation are well established, but where and when encapsidation occurs in the cell is unknown. We constructed MS2 phage coat protein labeling systems to track spatial dynamics of primate and nonprimate lentiviral genomic RNAs (HIV-1 and feline immunodeficiency virus [FIV]) vis-à-vis their Gag proteins in live cells. Genomic RNAs of both lentiviral genera were observed to traffic into the cytoplasm, and this was Rev dependent. In transit, FIV Gag and genomic RNA accumulated independently of each other at the nuclear envelope, and focal colocalizations of genomic RNA with an intact packaging signal (ψ) and Gag were observed to extend outward from the cytoplasmic face. In contrast, although HIV-1 genomic RNA was detected at the nuclear envelope, HIV-1 Gag was not. For both lentiviruses, genomic RNAs were seen at the plasma membrane if and only if Gag was present and ψ was intact. In addition, HIV-1 and FIV genomes accumulated with Gag in late endosomal foci, again, only ψ dependently. Thus, lentiviral genomic RNAs require specific Gag binding to accumulate at the plasma membrane, packaged genomes cointernalize with Gag into the endosomal pathway, and plasma membrane RNA incorporation by Gag does not trigger committed lentiviral particle egress from the cell. Based on the FIV results, we hypothesize that the Gag-genome association may initiate at the nuclear envelope.
The A20 Deubiquitinase Activity Negatively Regulates LMP1 Activation of IRF7
Authors: Ning Shunbin , Pagano Joseph S. 
Published: April 2010

ABSTRACT

A20 possesses both deubiquitinase (DUB) and ubiquitin E3 ligase activities that are required for termination of Toll-like receptor (TLR) signaling leading to NF-κB activation and for blockage of tumor necrosis factor (TNF)-induced cytotoxicity and apoptosis. A20 is induced by the Epstein-Barr virus (EBV) oncoprotein LMP1. However, its dual ubiquitin-editing activities have not been investigated in the context of either EBV infection or IRF7 responses. Both A20 and IRF7 have oncogenic properties. We have recently shown that LMP1 activates IRF7 through K63-linked ubiquitination which requires RIP1 and TRAF6, but how this ubiquitination event is regulated has not been studied. Here, we show that A20 negatively regulates IRF7 transcriptional activity induced by LMP1. Deletion or mutation of A20 C-terminal zinc finger motifs had no effect on the inhibition of IRF7 activity, whereas DUB-deficient truncation or point mutation ablated the ability of A20 to inhibit IRF7. Correspondingly, the A20 N-terminal DUB domain, but not the C-terminal E3 ligase domain, interacts physically with IRF7. Transient expression of A20 reduced K63-linked ubiquitination of IRF7
in vivo
, but an
in vitro
deubiquitination assay with purified constituents shows that IRF7 did not act as a substrate for A20 DUB activity. Moreover, A20 interacts with IRF7 endogenously in latently EBV-infected type 3 Raji cells, in which expression of both A20 and IRF7 is constitutively induced by the considerable level of endogenous LMP1. Knockdown of endogenous A20 in Raji cells by expression of A20 short hairpin RNA (shRNA) vectors increases endogenous IRF7 activity and ubiquitination, as well as the protein level of LMP1, a target of IRF7. Thus, A20 negatively regulates LMP1-stimulated IRF7 ubiquitination and activity in EBV latency, and its DUB activity is indispensable for this function. Finally, we discussed the regulation and function of IRFs in EBV latency.
DDX6 (Rck/p54) Is Required for Efficient Hepatitis C Virus Replication but Not for Internal Ribosome Entry Site-Directed Translation
Authors: Jangra Rohit K. , Yi MinKyung , Lemon Stanley M. 
Published: April 2010

ABSTRACT

DDX6 (Rck/p54) is an evolutionarily conserved member of the SF2 DEAD-box RNA helicase family that contributes to the regulation of translation and storage and the degradation of cellular mRNAs. It interacts with multiple proteins and is a component of the micro-RNA (miRNA)-induced silencing complex (miRISC). Since miRNA-122 (miR-122) is essential for efficient hepatitis C virus (HCV) replication, we investigated the requirement for DDX6 in HCV replication in cultured hepatoma cells. Small interfering RNA (siRNA)-mediated knockdown of DDX6 and rescue with an siRNA-resistant mutant demonstrated that DDX6 expression is indeed required for optimal HCV replication. However, DDX6 knockdown did not impair miR-122 biogenesis or alter HCV responsiveness to miR-122 supplementation. Overexpression of DDX6 fused to EYFP (EYFP-DDX6) enhanced replication, whereas a helicase-deficient mutant with a substitution in the conserved DEAD-box motif II (DQAD) had a dominant-negative effect, reducing HCV yields. Coimmunoprecipitation experiments revealed an intracellular complex containing DDX6, HCV core protein, and both viral and cellular RNAs, the formation of which was dependent upon the C-terminal domain of DDX6 but not DDX6 helicase activity. However, since DDX6 abundance influenced the replication of subgenomic HCV RNAs lacking core sequence, the relevance of this complex is uncertain. Importantly, DDX6 knockdown caused minimal reductions in cellular proliferation, generally stimulated cellular translation ([
35
S]Met incorporation), and did not impair translation directed by the HCV internal ribosome entry site. Thus, DDX6 helicase activity is essential for efficient HCV replication, reflecting essential roles for DDX6 in HCV genome amplification and/or maintenance of cellular homeostasis.
Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities
Authors: Tikhanovich Irina , Nasheuer Heinz Peter 
Published: April 2010

ABSTRACT
The activation of the human polyomavirus BK causes polyomavirus-associated nephropathy in immunocompromised humans. Studies of the virus have been restricted since the virus DNA replication is species specific. Cell-based and cell-free DNA replication systems, including the BK virus (BKV) monopolymerase DNA replication system using purified proteins, reproduce the species specificity (28). Therefore, the major host proteins comprising this assay, DNA polymerase α-primase (Pol-prim) and replication protein A (RPA), were intensively studied here. We demonstrate that Pol-prim plays a major role in the species specificity of BKV DNA replication. Both large subunits p180 and p68 of the enzyme complex have central functions in modulating the host specificity. Recently, an inhibitory activity of BKV DNA replication was described (C. Mahon, B. Liang, I. Tikhanovich, J. R. Abend, M. J. Imperiale, H. P. Nasheuer, and W. R. Folk, J. Virol. 83:5708-5717, 2009), but neither mouse Pol-prim nor mouse RPA diminishes cell-free BKV DNA replication. However, the inhibition of BKV DNA replication in mouse extracts depends on sequences flanking the core origin. In the presence of human Pol-prim, the inhibitory effect of mouse cell factors is abolished with plasmid DNAs containing the murine polyomavirus early promoter region, whereas the late enhancer region and the core origin are supplied from BKV. Thus, BKV replication is regulated by both Pol-prim, as a core origin species-specific factor, and inhibitory activities, as origin-flanking sequence-dependent factor(s).
Enhanced Gene Silencing in Cells Cured of Persistent Virus Infection by RNA Interference
Authors: Pelletier Isabelle , Saulnier Aure , Brisac Cynthia , Jegouic Sophie , Vabret Nicolas , Tangy Frédéric , Blondel Bruno , Colbère-Garapin Florence 
Published: April 2010

ABSTRACT
We compared HEp-2-derived cells cured of persistent poliovirus infection by RNA interference (RNAi) with parental cells, to investigate possible changes in the efficiency of RNAi. Lower levels of poliovirus replication were observed in cured cells, possibly facilitating virus silencing by antiviral small interfering RNAs (siRNAs). However, green fluorescent protein (GFP) produced from a measles virus vector and also GFP and luciferase produced from plasmids that do not replicate in human cells were more effectively silenced by specific siRNAs in cured than in control cells. Thus, cells displaying enhanced silencing were selected during curing by RNAi. Our results strongly suggest that the RNAi machinery of cured cells is more efficient than that of parental cells.
RASCAL Is a New Human Cytomegalovirus-Encoded Protein That Localizes to the Nuclear Lamina and in Cytoplasmic Vesicles at Late Times Postinfection
Authors: Miller Matthew S. , Furlong Wendy E. , Pennell Leesa , Geadah Marc , Hertel Laura 
Published: April 2010

ABSTRACT

The products of numerous open reading frames (ORFs) present in the genome of human cytomegalovirus (CMV) have not been characterized. Here, we describe the identification of a new CMV protein localizing to the nuclear envelope and in cytoplasmic vesicles at late times postinfection. Based on this distinctive localization pattern, we called this new protein nuclear

r

im-

as

sociated

c

ytomegalovir

al

protein, or RASCAL. Two RASCAL isoforms exist, a short version of 97 amino acids encoded by the majority of CMV strains and a longer version of 176 amino acids encoded by the Towne, Toledo, HAN20, and HAN38 strains. Both isoforms colocalize with lamin B in deep intranuclear invaginations of the inner nuclear membrane (INM) and in novel cytoplasmic vesicular structures possibly derived from the nuclear envelope. INM infoldings have been previously described as sites of nucleocapsid egress, which is mediated by the localized disruption of the nuclear lamina, promoted by the activities of viral and cellular kinases recruited by the lamina-associated proteins UL50 and UL53. RASCAL accumulation at the nuclear membrane required the presence of UL50 but not of UL53. RASCAL and UL50 also appeared to specifically interact, suggesting that RASCAL is a new component of the nuclear egress complex (NEC) and possibly involved in mediating nucleocapsid egress from the nucleus. Finally, the presence of RASCAL within cytoplasmic vesicles raises the intriguing possibility that this protein might participate in additional steps of virion maturation occurring after capsid release from the nucleus.
Antibody-Mediated Protection against Mucosal Simian-Human Immunodeficiency Virus Challenge of Macaques Immunized with Alphavirus Replicon Particles and Boosted with Trimeric Envelope Glycoprotein in MF59 Adjuvant
Authors: Barnett Susan W. , Burke Brian , Sun Yide , Kan Elaine , Legg Harold , Lian Ying , Bost Kristen , Zhou Fengmin , Goodsell Amanda , zur Megede Jan , Polo John , Donnelly John , Ulmer Jeffrey , Otten Gillis R. , Miller Christopher J. , Vajdy Michael , Srivastava Indresh K. 
Published: April 2010

ABSTRACT

We have previously shown that rhesus macaques were partially protected against high-dose intravenous challenge with simian-human immunodeficiency virus SHIV
SF162P4
following sequential immunization with alpha
v
irus
r
eplicon
p
articles (VRP) of a chimeric recombinant VEE/SIN alphavirus (derived from Venezuelan equine encephalitis virus [VEE] and the Sindbis virus [SIN]) encoding human immunodeficiency virus type 1 HIV-1
SF162
gp140ΔV2 envelope (Env) and trimeric Env protein in MF59 adjuvant (R. Xu, I. K. Srivastava, C. E. Greer, I. Zarkikh, Z. Kraft, L. Kuller, J. M. Polo, S. W. Barnett, and L. Stamatatos, AIDS Res. Hum. Retroviruses 22:1022-1030, 2006). The protection did not require T-cell immune responses directed toward simian immunodeficiency virus (SIV) Gag. We extend those findings here to demonstrate antibody-mediated protection against mucosal challenge in macaques using prime-boost regimens incorporating both intramuscular and mucosal routes of delivery. The macaques in the vaccination groups were primed with VRP and then boosted with Env protein in MF59 adjuvant, or they were given VRP intramuscular immunizations alone and then challenged with SHIV
SF162P4
(intrarectal challenge). The results demonstrated that these vaccines were able to effectively protect the macaques to different degrees against subsequent mucosal SHIV challenge, but most noteworthy, all macaques that received the intramuscular VRP prime plus Env protein boost were completely protected. A statistically significant association was observed between the titer of virus neutralizing and binding antibodies as well as the avidity of anti-Env antibodies measured prechallenge and protection from infection. These results highlight the merit of the alphavirus replicon vector prime plus Env protein boost vaccine approach for the induction of protective antibody responses and are of particular relevance to advancing our understanding of the potential correlates of immune protection against HIV infection at a relevant mucosal portal of entry.
2A Protease Is Not a Prerequisite for Poliovirus Replication
Authors: Igarashi Hiroko , Yoshino Yasuko , Miyazawa Miwako , Horie Hitoshi , Ohka Seii , Nomoto Akio 
Published: April 2010

ABSTRACT

Poliovirus (PV) 2A
pro
has been considered important for PV replication and is known to be toxic to host cells. A 2A
pro
-deficient PV would potentially be less toxic and ideal as a vector. To examine whether 2A
pro
is needed to form progeny virus, a full-length cDNA of dicistronic (
dc
) PV with (pOME) or without (pOMEΔ2A) 2A
pro
was constructed in the strain PV1(M)OM. RNAs of both pOME and pOMEΔ2A were capable of forming progeny viruses, called OME and OMEΔ2A, respectively. In their ability to induce a cytopathic effect (CPE), the strains ranked as OMEΔ2A < OME ≒ PV1(M)OM. These results suggest that 2A
pro
is not essential for full-length
dc
PV to form progeny virus and that it contributes to the efficient viral replication and/or induction of a CPE. To clarify whether 2A
pro
is essential for P1-null (lacking the entire coding sequence for capsid proteins) PV, the RNA replication activity of P1-null PV (pOMΔP1) or P1-null PV without 2A
pro
(pOMΔP1Δ2A) or without both 2A
pro
and 2B (pOMΔP1Δ2AΔ2B) was examined. The RNAs of pOMΔP1 and pOMΔP1Δ2A could replicate and form progeny viruses under a
trans
supply of P1 protein, whereas the RNA of pOMΔP1Δ2AΔ2B could not. These results suggest that 2A
pro
is not needed for the replication of P1-null PV, although it is important for PV RNA replication and inducing a CPE. To know whether a 2A
pro
-deficient PV can be used as a vector, a P1-null PV containing the enhanced green fluorescent protein (EGFP) coding sequence with or without 2A
pro
was examined. It expressed fluorescent protein. This result suggests that 2A
pro
-deficient PV can express foreign genes.
In Vitro
Assessment of Attachment Pattern and Replication Efficiency of H5N1 Influenza A Viruses with Altered Receptor Specificity
Authors: Chutinimitkul Salin , van Riel Debby , Munster Vincent J. , van den Brand Judith M. A. , Rimmelzwaan Guus F. , Kuiken Thijs , Osterhaus Albert D. M. E. , Fouchier Ron A. M. , de Wit Emmie 
Published: April 2010

ABSTRACT
The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, α2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to α2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with α2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.