bluuebunny/crossref_metadata_2025 huggingface dataset:
Fosfomycin Suppresses Chemokine Induction in Airway Epithelial Cells Infected with Respiratory Syncytial Virus
Authors: Okabayashi Tamaki , Yokota Shin-ichi , Yoto Yuko , Tsutsumi Hiroyuki , Fujii Nobuhiro
Published: April 2009

Respiratory syncytial virus (RSV) infects airway epithelial cells, causing bronchiolitis and pneumonia. Inflammation is mediated by various cytokines secreted from RSV-infected airway epithelial cells, and it promotes the pathogenesis of RSV-related diseases. Fosfomycin (FOF) is approved as a treatment for various bacterial infectious diseases, including respiratory infectious diseases, in Japan. FOF is suggested to exhibit immunomodulatory effects on lipopolysaccharide-stimulated monocytes and T lymphocytes, in addition to its antimicrobial activity. We investigated the effect of FOF on the cytokine production of an airway epithelial cell line, A549, infected with RSV. RSV-induced cytokines, such as regulated on activation, normal T-cell expressed and secreted (RANTES), interleukin-8 (IL-8), and IL-6, in infected A549 cells. We found that FOF decreased the levels of RSV-induced RANTES and IL-8 but not the level of RSV-induced IL-6. The RANTES promoter was activated by RSV infection. Site-directed mutagenesis analysis of the RANTES promoter showed that NF-κB-binding motifs had a critical role in RSV-induced RANTES promoter activity. A luciferase reporter gene assay and a DNA-binding assay indicated that FOF suppressed the NF-κB activity induced by RSV infection. These results demonstrate that FOF treatment suppresses the RSV-induced transcription of the chemokines RANTES and IL-8 in airway epithelial cells.
Expression of Interleukin-5 and Tumor Necrosis Factor Alpha in Cervical Carcinoma
Authors: Lee Dae Woo , Kim Young Tae , Kim Sang Wun , Kim SungHoon , Kim Jae Hoon , Kang Myung Hwa , Hwang Jeong Hye , Lim Jong baeck
Published: April 2009

Interleukin-5 (IL-5) levels were significantly higher in vaginal washing fluids from patients with cervical carcinoma than in those from patients with carcinoma in situ and controls. Tumor necrosis factor alpha levels did not differ among the three groups. Detection of IL-5 in cervical secretions may be a useful marker for evaluating aggressive local immune response in cervical carcinoma.
Antibodies to
Staphylococcus aureus
Bone Sialoprotein-Binding Protein Indicate Infectious Osteomyelitis
Authors: Persson Lena , Johansson Christian , Rydén Cecilia
Published: April 2009

Discrimination of soft tissue infection from osteomyelitis in diabetic foot infections is a common clinical problem.
Staphylococcus aureus
isolates from patients with osteomyelitis express bone sialoprotein-binding protein (Bbp) that binds the bone matrix protein bone sialoprotein. The serological assay with Bbp discriminated cases of osteomyelitis from soft tissue infections in patients with diabetic foot ulcers.
Development and Evaluation of a Sensitive and Specific Assay for Diagnosis of Human Toxocariasis by Use of Three Recombinant Antigens (TES-26, TES-30USM, and TES-120)
Authors: Mohamad Suharni , Azmi Norhaida Che , Noordin Rahmah
Published: April 2009

Diagnosis of human toxocariasis currently relies on serologic tests that useToxocaraexcretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathioneS-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed inEscherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis.
Isolation of
Bartonella vinsonii
subsp.
berkhoffii
Genotype II from a Boy with Epithelioid Hemangioendothelioma and a Dog with Hemangiopericytoma
Authors: Breitschwerdt Edward B. , Maggi Ricardo G. , Varanat Mrudula , Linder Keith E. , Weinberg Guy
Published: April 2009

In this report, we describe isolation of
Bartonella vinsonii
subsp.
berkhoffii
genotype II from a boy with epithelioid hemangioendothelioma and a dog with hemangiopericytoma. These results suggest that
B
.
vinsonii
subsp.
berkhoffii
may cause vasoproliferative lesions in both humans and dogs.
Comparison of the Bactec 9240 and BacT/Alert Blood Culture Systems for Evaluation of Placental Cord Blood for Transfusion in Neonates
Authors: Riedel Stefan , Junkins Alan , Stamper Paul D. , Cress Gretchen , Widness John A. , Doern Gary V.
Published: April 2009

The Bactec 9240 and the BacT/Alert blood culture systems were compared as a means for detection of bacterial contaminants in whole blood, concentrated red cells, and plasma preparations prepared from umbilical cord blood (UCB) samples. Ninety-two UCB units seeded with low levels of various bacteria were evaluated. In more than 50% of cases, growth was not detected in plasma using either system (
P
< 0.001). When concentrated red cells and whole blood were compared, the Bactec system detected bacterial growth consistently sooner than the BacT/Alert system in all seeded bacteria except
Staphylococcus
species in whole blood. The median lengths of time to detection (LTD) for whole blood and concentrated cells in BacT/Alert were 18.7 h and 18.5 h, respectively. The median LTD for the same blood fractions using the Bactec system were 16.05 h and 15.64 h. These differences in LTD by blood culture system and sample type were statistically significant (whole blood,
P
= 0.0449; concentrated cells,
P
= 0.0037). Based on the results of our study, we recommend the use of either concentrated red cells or whole blood for sterility testing in UCB samples. In our laboratory, the Bactec system compared to the BacT/Alert system was the superior method for rapid detection of bacterial contaminants in cord blood.
First Report of
Tsukamurella
Keratitis: Association between
T. tyrosinosolvens
and
T. pulmonis
and Ophthalmologic Infections
Authors: Woo Patrick C. Y. , Fong Angie H. C. , Ngan Antonio H. Y. , Tam Dorothy M. W. , Teng Jade L. L. , Lau Susanna K. P. , Yuen Kwok-Yung
Published: April 2009

We describe the first two cases of
Tsukamurella
keratitis, presented as eye pain with or without blurring of vision. One case was associated with trichiasis and the other with contact lens wear. The two isolates were identified as
T. tyrosinosolvens
and
T. pulmonis
, respectively, by phenotypic characterization and 16S rRNA sequencing.
Peptide Nucleic Acid-Based Array for Detecting and Genotyping Human Papillomaviruses
Authors: Choi Jae-jin , Kim Chunhee , Park Heekyung
Published: April 2009

We describe a novel array for accurate and reliable genotyping of human papillomavirus (HPV) using peptide nucleic acid (PNA) probes. In order to exploit the superior hybridization properties of PNA with target HPV DNAs, we developed a novel PNA array (PANArray HPV). PANArray HPV enables the detection and genotyping of HPVs using 32 type-specific PNA capture probes for medically important HPVs. All tested HPV types showed highly unique hybridization patterns with type-specific PNA probes. PNA array results showed stable specificities and sensitivities after up to 13 months of storage at room temperature. Also, we demonstrated the superior specificity, sensitivity, and stability of PNA arrays for HPV genotyping. We compared the genotyping results of the PNA array to sequencing with MY09/11 PCR products derived from 72 clinical samples. The results showed excellent agreement between the PNA array and sequencing, except for samples reflecting multiple infections. The results from the PNA array were compared with those of type-specific PCR when discrepant results occurred owing to multiple infections. The results for the PNA array matched those of type-specific PCR in all cases. Newly developed PNA arrays show excellent specificity and sensitivity and long shelf life. Our results suggest that the PNA array represents a reliable alternative to conventional DNA arrays for HPV genotyping, as well as for diagnostics.
Ureaplasma parvum
as a Cause of Sternal Wound Infection
Authors: Walkty Andrew , Lo Evelyn , Manickam Kanchana , Alfa Michelle , Xiao Li , Waites Ken
Published: April 2009

Few reports in the literature have documented the isolation of
Ureaplasma
species from sternal wounds. A case of sternal wound infection likely due to
Ureaplasma parvum
is described. When routine bacterial cultures from a sternal wound infection fail to yield a pathogen, diagnostic testing for mycoplasmas and ureaplasmas should be considered.
Evaluation of the BD GeneOhm StaphSR Assay for Detection of Methicillin-Resistant and Methicillin-Susceptible
Staphylococcus aureus
Isolates from Spiked Positive Blood Culture Bottles
Authors: Gröbner Sabine , Dion Mireille , Plante Mélanie , Kempf Volkhard A. J.
Published: April 2009

To improve the clinical outcome of
Staphylococcus aureus
septicemia, the early selection of appropriate antibiotic treatment is crucial. Molecular diagnostics represents an attractive approach for the rapid identification of
S. aureus
and the determination of its methicillin (meticillin) resistance. In direct comparison to other molecular assays (
sa442
and
mecA
real-time PCRs) and standard laboratory procedures, we evaluated the BD GeneOhm StaphSR assay for its use in the detection of
S. aureus
and methicillin-resistant
S. aureus
(MRSA) from spiked blood culture bottles (
n
= 134). In the case of detecting
S. aureus
(
n
= 90; for methicillin-susceptible
S. aureus
,
n
= 45; for MRSA,
n
= 45), the BD GeneOhm StaphSR assay had a sensitivity and a specificity of 100% each (95% confidence intervals [CIs], 96.0 to 100% and 82.4 to 100%, respectively). For MRSA (
n
= 45), the test was 95.6% (95% CI, 84.9 to 99.5%) sensitive and 95.3% (95% CI, 86.9 to 99.0%) specific. Overall, five discrepant results arose with this assay due to the presence of methicillin-susceptible, revertant MRSA strains (3/45) and MRSA strains that were not detected by the BD GeneOhm StaphSR assay (2/45). Compared to other real-time PCR-based molecular approaches and to conventional standard laboratory methods, the BD GeneOhm StaphSR turned out to be an appropriate diagnostic tool for a rapid (∼1.5 h), preliminary identification of
S. aureus
and MRSA from blood cultures.
Cell Cycle Control of Kaposi's Sarcoma-Associated Herpesvirus Latency Transcription by CTCF-Cohesin Interactions
Authors: Kang Hyojeung , Lieberman Paul M.
Published: April 2009

Kaposi's sarcoma-associated herpesvirus (KSHV) latency is characterized by the highly regulated transcription of a few viral genes essential for genome maintenance and host cell survival. A major latency control region has been identified upstream of the divergent promoters for the multicistronic transcripts encoding LANA (ORF73), vCyclin (ORF72), and vFLIP (ORF71) and for the complementary strand transcript encoding K14 and vGPCR (ORF74). Previous studies have shown that this major latency control region is occupied by the cellular chromatin boundary factor CTCF and chromosome structural maintenance proteins SMC1, SMC3, and RAD21, which comprise the cohesin complex. Deletion of the CTCF-cohesin binding site caused an inhibition of cell growth and viral genome instability. We now show that the KSHV genes regulated by CTCF-cohesin are under cell cycle control and that mutation of the CTCF binding sites abolished cell cycle-regulated transcription. Cohesin subunits assembled at the CTCF binding sites and bound CTCF proteins in a cell cycle-dependent manner. Subcellular distribution of CTCF and colocalization with cohesins also varied across the cell cycle. Ectopic expression of Rad21 repressed CTCF-regulated transcription of KSHV lytic genes, and a Rad21-CTCF chimeric protein converted CTCF into an efficient transcriptional repressor of KSHV genes normally activated in the G
2
phase. We conclude that cohesins interact with CTCF in mid-S phase and repress CTCF-regulated genes in a cell cycle-dependent manner. We propose that the CTCF-cohesin complex plays a critical role in regulating the cell cycle control of viral gene expression during latency and that failure to maintain cell cycle control of latent transcripts inhibits host cell proliferation and survival.
Breaking Human Cytomegalovirus Major Immediate-Early Gene Silence by Vasoactive Intestinal Peptide Stimulation of the Protein Kinase A-CREB-TORC2 Signaling Cascade in Human Pluripotent Embryonal NTera2 Cells
Authors: Yuan Jinxiang , Liu Xiaoqiu , Wu Allen W. , McGonagill Patrick W. , Keller Michael J. , Galle Courtney S. , Meier Jeffery L.
Published: April 2009

The triggering mechanisms underlying reactivation of human cytomegalovirus (HCMV) in latently infected persons are unclear. During latency, HCMV major immediate-early (MIE) gene expression breaks silence to initiate viral reactivation. Using quiescently HCMV-infected human pluripotent embryonal NTera2 cells (NT2) to model HCMV reactivation, we show that vasoactive intestinal peptide (VIP), an immunomodulatory neuropeptide, immediately and dose-dependently (1 to 500 nM) activates HCMV MIE gene expression. This response requires the MIE enhancer cyclic AMP response elements (CRE). VIP quickly elevates CREB Ser133 and ATF-1 Ser63 phosphorylation levels, although the CREB Ser133 phosphorylation level is substantial at baseline. VIP does not change the level of HCMV genomes in nuclei, Oct4 (pluripotent cell marker), or hDaxx (cellular repressor of HCMV gene expression). VIP-activated MIE gene expression is mediated by cellular protein kinase A (PKA), CREB, and TORC2. VIP induces PKA-dependent TORC2 Ser171 dephosphorylation and nuclear entry, which likely enables MIE gene activation, as TORC2 S171A (devoid of Ser171 phosphorylation) exhibits enhanced nuclear entry and desilences the MIE genes in the absence of VIP stimulation. In conclusion, VIP stimulation of the PKA-CREB-TORC2 signaling cascade activates HCMV CRE-dependent MIE gene expression in quiescently infected NT2 cells. We speculate that neurohormonal stimulation via this signaling cascade is a possible means for reversing HCMV silence in vivo.
Herpes Simplex Virus Type 1 Suppresses RNA-Induced Gene Silencing in Mammalian Cells
Authors: Wu Zetang , Zhu Yali , Bisaro David M. , Parris Deborah S.
Published: April 2009

RNA-induced silencing is a potent innate antiviral defense strategy in plants, and suppression of silencing is a hallmark of pathogenic plant viruses. However, the impact of silencing as a mammalian antiviral defense mechanism and the ability of mammalian viruses to suppress silencing in natural host cells have remained controversial. The ability of herpes simplex virus type 1 (HSV-1) to suppress silencing was examined in a transient expression system that employed an imperfect hairpin to target degradation of transcripts encoding enhanced green fluorescent protein (EGFP). HSV-1 infection suppressed EGFP-specific silencing as demonstrated by increased EGFP mRNA levels and an increase in the EGFP mRNA half-life. The increase in EGFP mRNA stability occurred despite the well-characterized host macromolecular shutoff functions of HSV-1 that globally destabilize mRNAs. Moreover, mutant viruses defective in these functions increased the stability of EGFP mRNA even more than did the wild-type virus in silenced cells compared to results in control cells. The importance of RNA silencing to HSV-1 replication was confirmed by a significantly enhanced virus burst size in cells in which silencing was knocked down with small inhibitory RNAs directed to Argonaute 2, an integral component of the silencing complex. Given that HSV-1 encodes several microRNAs, it is possible that a dynamic equilibrium exists between silencing and silencing suppression that is capable of modulating viral gene expression to promote replication, to evade host defenses, and/or to promote latency.
Cytotoxic-T-Lymphocyte-Mediated Elimination of Target Cells Transduced with Engineered Adeno-Associated Virus Type 2 Vector In Vivo
Authors: Li Chengwen , Hirsch Matt , DiPrimio Nina , Asokan Aravind , Goudy Kevin , Tisch Roland , Samulski R. Jude
Published: April 2009

A recent clinical trial in patients with hemophilia B has suggested that adeno-associated virus (AAV) capsid-specific cytotoxic T lymphocytes (CTLs) eliminated AAV-transduced hepatocytes and resulted in therapeutic failure. AAV capsids elicit a CTL response in animal models; however, these capsid-specific CTLs fail to kill AAV-transduced target cells in mice. To better model the human clinical trial data in mice, we introduced an immunodominant epitope derived from ovalbumin (OVA; SIINFEKL) into the AAV capsid and tested CTL-mediated killing of AAV2-transduced target tissues in vivo. Initially, in vitro experiments demonstrated both classical class I and cross-presentation of the OVA antigen, following endogenous expression or AAV2-OVA vector transduction, respectively. Furthermore, an OVA-specific CTL response was elicited after muscular or systemic injection of the AAV2-OVA vector. Finally, CTL reactivity was enhanced in mice with established SIINFEKL-specific immunity after AAV2-OVA/α1 anti-trypsin (AAT) administration. Most importantly, these OVA-specific CTLs decreased AAT expression in mice treated with AAV2-OVA/AAT vector that followed a time course mimicking uncoating kinetics of AAV2 transduction in OVA-immunized mice. These results demonstrate that AAV capsid-derived antigens elicit CD8
+
CTL reactivity, and these CTLs eliminated AAV-transduced target cells in mice. Notably, this model system can be exploited to study the kinetics of capsid presentation from different serotypes of AAV and permit the design of novel strategies to block CTL-mediated killing of AAV-transduced cells.
Herpes Simplex Virus Glycoprotein B Associates with Target Membranes via Its Fusion Loops
Authors: Hannah Brian P. , Cairns Tina M. , Bender Florent C. , Whitbeck J. Charles , Lou Huan , Eisenberg Roselyn J. , Cohen Gary H.
Published: April 2009

Herpes simplex virus (HSV) glycoproteins gB, gD, and gH/gL are necessary and sufficient for virus entry into cells. Structural features of gB are similar to those of vesicular stomatitis virus G and baculovirus gp64, and together they define the new class III group of fusion proteins. Previously, we used mutagenesis to show that three hydrophobic residues (W174, Y179, and A261) within the putative gB fusion loops are integral to gB function. Here we expanded our analysis, using site-directed mutagenesis of each residue in both gB fusion loops. Mutation of most of the nonpolar or hydrophobic amino acids (W174, F175, G176, Y179, and A261) had severe effects on gB function in cell-cell fusion and null virus complementation assays. Of the six charged amino acids, mutation of H263 or R264 also negatively affected gB function. To further analyze the mutants, we cloned the ectodomains of the W174R, Y179S, H263A, and R264A mutants into a baculovirus expression system and compared them with the wild-type (WT) form, gB730t. As shown previously, gB730t blocks virus entry into cells, suggesting that gB730t competes with virion gB for a cell receptor. All four mutant proteins retained this function, implying that fusion loop activity is separate from gB-receptor binding. However, unlike WT gB730t, the mutant proteins displayed reduced binding to cells and were either impaired or unable to bind naked, cholesterol-enriched liposomes, suggesting that it may be gB-lipid binding that is disrupted by the mutations. Furthermore, monoclonal antibodies with epitopes proximal to the fusion loops abrogated gB-liposome binding. Taken together, our data suggest that gB associates with lipid membranes via a fusion domain of key hydrophobic and hydrophilic residues and that this domain associates with lipid membranes during fusion.
Kaposi's Sarcoma-Associated Herpesvirus RTA Promotes Degradation of the Hey1 Repressor Protein through the Ubiquitin Proteasome Pathway
Authors: Gould Faye , Harrison Sally M. , Hewitt Eric W. , Whitehouse Adrian
Published: April 2009

The Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional repressor proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional repressor protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the corepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation.
Human Immunodeficiency Virus Integration Efficiency and Site Selection in Quiescent CD4
+
T Cells
Authors: Vatakis Dimitrios N. , Kim Sanggu , Kim Namshin , Chow Samson A. , Zack Jerome A.
Published: April 2009

Until very recently, quiescent CD4
+
T cells were thought to be resistant to human immunodeficiency virus (HIV) infection. Subsequent studies, attempting to fully elucidate the mechanisms of resistance, showed that quiescent cells could become infected by HIV at low efficiency and form a latently infected population. In this study, we set out to identify the sites of viral integration and to assess the efficiency of the overall integration process in quiescent cells. Based on our results, HIV integration in quiescent CD4
+
T cells occurs in sites similar to those of their prestimulated counterparts. While site selections are similar, the integration process in quiescent cells is plagued by the formation of high levels of incorrectly processed viral ends and abortive two-long-terminal-repeat circles.
The Cellular RNA Export Receptor TAP/NXF1 Is Required for ICP27-Mediated Export of Herpes Simplex Virus 1 RNA, but the TREX Complex Adaptor Protein Aly/REF Appears To Be Dispensable
Authors: Johnson Lisa A. , Li Ling , Sandri-Goldin Rozanne M.
Published: April 2009

Herpes simplex virus 1 (HSV-1) protein ICP27 has been shown to shuttle between the nucleus and cytoplasm and to bind viral RNA during infection. ICP27 was found to interact with the cellular RNA export adaptor protein Aly/REF, which is part of the TREX complex, and to relocalize Aly/REF to viral replication sites. ICP27 is exported to the cytoplasm through the export receptor TAP/NXF1, and ICP27 must be able to interact with TAP/NXF1 for efficient export of HSV-1 early and late transcripts. We examined the dynamics of ICP27 movement and its localization with respect to Aly/REF and TAP/NXF1 in living cells during viral infection. Recombinant viruses with a yellow fluorescent protein (YFP) tag on the N or C terminus of ICP27 were constructed. While the N-terminally tagged ICP27 virus behaved like wild-type HSV-1, the C-terminally tagged virus was defective in viral replication and gene expression, and ICP27 was confined to the nucleus, suggesting that the C-terminal YFP tag interfered with ICP27's C-terminal interactions, including the interaction with TAP/NXF1. To assess the role of Aly/REF and TAP/NXF1 in viral RNA export, these factors were knocked down using small interfering RNA. Knockdown of Aly/REF had little effect on the export of ICP27 or poly(A)
+
RNA during infection. In contrast, a decrease in TAP/NXF1 levels severely impaired export of ICP27 and poly(A)
+
RNA. We conclude that TAP/NXF1 is essential for ICP27-mediated export of RNA during HSV-1 infection, whereas Aly/REF may be dispensable.
Kinetic Dependence of Paramyxovirus Entry Inhibition
Authors: Porotto Matteo , Yokoyama Christine C. , Orefice Gianmarco , Kim Han-Sung , Aljofan Mohamed , Mungall Bruce A. , Moscona Anne
Published: April 2009

Peptides derived from conserved heptad repeat (HR) regions of paramyxovirus fusion (F) proteins inhibit viral fusion by interfering with the formation of the fusogenic six-helix bundle structure. Peptide efficacy is affected by the strength of the peptide association with the target virus's complementary HR region. Here, we show that a second basis for peptide efficacy lies in the kinetics of F activation by the homotypic attachment protein: efficient F activation by the attachment protein shortens the period during which antiviral molecules targeting intermediate states of F may act, thereby modulating the effectiveness of inhibitory peptides. These results highlight new issues to be considered in developing strategies for fusion inhibitors.
Identification of Cellular Genes Affecting the Infectivity of Foot-and-Mouth Disease Virus
Authors: Piccone Maria E. , Feng Yanan , Chang Annie C. Y. , Mosseri Ronen , Lu Quan , Kutish Gerald F. , Lu Zhiqiang , Burrage Thomas G. , Gooch Christina , Rock Daniel L. , Cohen Stanley N.
Published: April 2009

Foot-and-mouth disease virus (FMDV) produces one of the most infectious of all livestock diseases, causing extensive economic loss in areas of breakout. Like other viral pathogens, FMDV recruits proteins encoded by host cell genes to accomplish the entry, replication, and release of infectious viral particles. To identify such host-encoded proteins, we employed an antisense RNA strategy and a lentivirus-based library containing approximately 40,000 human expressed sequence tags (ESTs) to randomly inactivate chromosomal genes in a bovine kidney cell line (LF-BK) that is highly susceptible to FMDV infection and then isolated clones that survived multiple rounds of exposure to the virus. Here, we report the identification of ESTs whose expression in antisense orientation limited host cell killing by FMDV and restricted viral propagation. The role of one such EST, that of ectonucleoside triphosphate diphosphohydrolase 6 (NTPDase6; also known as CD39L2), a membrane-associated ectonucleoside triphosphate diphosphohydrolase that previously was not suspected of involvement in the propagation of viral pathogens and which we now show is required for normal synthesis of FMDV RNA and proteins, is described in this report.
Role of Microtubules in Extracellular Release of Poliovirus
Authors: Taylor Matthew P. , Burgon Trever B. , Kirkegaard Karla , Jackson William T.
Published: April 2009

Cellular autophagy, a process that directs cytosolic contents to the endosomal and lysosomal pathways via the formation of double-membraned vesicles, is a crucial aspect of innate immunity to many intracellular pathogens. However, evidence is accumulating that certain RNA viruses, such as poliovirus, subvert this pathway to facilitate viral growth. The autophagosome-like membranes induced during infection with wild-type poliovirus were found to be, unlike cellular autophagosomes, relatively immobile. Their mobility increased upon nocodazole treatment, arguing that vesicular tethering is microtubule dependent. In cells infected with a mutant virus that is defective in its interaction with the host cytoskeleton and secretory pathway, vesicle movement increased, indicating reduced tethering. In all cases, the release of tethering correlated with increased amounts of extracellular virus, which is consistent with the hypothesis that small amounts of cytosol and virus entrapped by double-membraned structures could be released via fusion with the plasma membrane. We propose that this extracellular delivery of cytoplasmic contents be termed autophagosome-mediated exit without lysis (AWOL). This pathway could explain the observed exit, in the apparent absence of cellular lysis, of other cytoplasmic macromolecular complexes, including infectious agents and complexes of aggregated proteins.
Integrase Interacts with Nucleoporin NUP153 To Mediate the Nuclear Import of Human Immunodeficiency Virus Type 1
Authors: Woodward Cora L. , Prakobwanakit Sarin , Mosessian Sherly , Chow Samson A.
Published: April 2009

The ability to traverse an intact nuclear envelope and productively infect nondividing cells is a salient feature of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses, but the viral factors and mechanism of nuclear entry have not been defined. HIV-1 integrase (IN) is implicated to play a role in the nuclear import of the virus, but the cellular pathway for IN trafficking and the role of IN in mediating the nuclear import of viral particles are unknown. Using a semipermeabilized cell assay, we observed that the nuclear import of IN was not the result of passive diffusion but occurred independently of cytosolic factors, metabolic energy, and the classical receptor-mediated, Ran-dependent import pathways. To determine if IN enters the nucleus by interacting with the nucleopore complex (NPC), we found that IN bound directly with the FxFG-rich C-terminal domain of nucleoporin 153 (NUP153C). When added in excess to the import assay, NUP153C inhibited the nuclear import of IN. Known binding partners of NUP153C competed with IN for binding with NUP153 and also inhibited the nuclear import of IN. In cultured cells, overexpression of NUP153C reduced the infectivity of an HIV-derived vector by interfering with the nuclear translocation of the viral cDNA. These results support a functional role for the IN-NUP153 interaction in HIV-1 replication and suggest that HIV-1 subviral particles gain access to the nucleus by interacting directly with the NPC via the binding of particle-associated IN to NUP153C.
Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease Ubiquitin-Like Domain and Catalytic Domain Regulate Antagonism of IRF3 and NF-κB Signaling
Authors: Frieman Matthew , Ratia Kiira , Johnston Robert E. , Mesecar Andrew D. , Baric Ralph S.
Published: April 2009

The outcome of a viral infection is regulated in part by the complex coordination of viral and host interactions that compete for the control and optimization of virus replication. Severe acute respiratory syndrome coronavirus (SARS-CoV) intimately engages and regulates the host innate immune responses during infection. Using a novel interferon (IFN) antagonism screen, we show that the SARS-CoV proteome contains several replicase, structural, and accessory proteins that antagonize the IFN pathway. In this study, we focus on the SARS-CoV papain-like protease (PLP), which engages and antagonizes the IFN induction and NF-κB signaling pathways. PLP blocks these pathways by affecting activation of the important signaling proteins in each pathway, IRF3 and NF-κB. We also show that the ubiquitin-like domain of PLP is necessary for pathway antagonism but not sufficient by itself to block these pathways regardless of the enzymatic activity of the protease. The potential mechanism of PLP antagonism and its role in pathogenesis are discussed.
Dissecting the Functional Domains of a Nonenveloped Virus Membrane Penetration Peptide
Authors: Banerjee Manidipa , Khayat Reza , Walukiewicz Hanna E. , Odegard Amy L. , Schneemann Anette , Johnson John E.
Published: April 2009

Recent studies have established that several nonenveloped viruses utilize virus-encoded lytic peptides for host membrane disruption. We investigated this mechanism with the “gamma” peptide of the insect virus Flock House virus (FHV). We demonstrate that the C terminus of gamma is essential for membrane disruption in vitro and the rescue of immature virus infectivity in vivo, and the amphipathic N terminus of gamma alone is not sufficient. We also show that deletion of the C-terminal domain disrupts icosahedral ordering of the amphipathic helices of gamma in the virus. Our results have broad implications for understanding membrane lysis during nonenveloped virus entry.
Identification of a Feline Leukemia Virus Variant That Can Use THTR1, FLVCR1, and FLVCR2 for Infection
Authors: Shalev Zvi , Duffy Simon P. , Adema Karen W. , Prasad Rati , Hussain Naveen , Willett Brian J. , Tailor Chetankumar S.
Published: April 2009

The pathogenic subgroup C feline leukemia virus (FeLV-C) arises in infected cats as a result of mutations in the envelope (Env) of the subgroup A FeLV (FeLV-A). To better understand emergence of FeLV-C and potential FeLV intermediates that may arise, we characterized FeLV Env sequences from the primary FY981 FeLV isolate previously derived from an anemic cat. Here, we report the characterization of the novel FY981 FeLV Env that is highly related to FeLV-A Env but whose variable region A (VRA) receptor recognition sequence partially resembles the VRA sequence from the prototypical FeLV-C/Sarma Env. Pseudotype viruses bearing FY981 Env were capable of infecting feline, human, and guinea pig cells, suggestive of a subgroup C phenotype, but also infected porcine ST-IOWA cells that are normally resistant to FeLV-C and to FeLV-A. Analysis of the host receptor used by FY981 suggests that FY981 can use both the FeLV-C receptor FLVCR1 and the feline FeLV-A receptor THTR1 for infection. However, our results suggest that FY981 infection of ST-IOWA cells is not mediated by the porcine homologue of FLVCR1 and THTR1 but by an alternative receptor, which we have now identified as the FLVCR1-related protein FLVCR2. Together, our results suggest that FY981 FeLV uses FLVCR1, FLVCR2, and THTR1 as receptors. Our findings suggest the possibility that pathogenic FeLV-C arises in FeLV-infected cats through intermediates that are multitropic in their receptor use.
Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 Targets MDM2 To Deregulate the p53 Tumor Suppressor Pathway
Authors: Lee Hye-Ra , Toth Zsolt , Shin Young C. , Lee Jong-Soo , Chang Heesoon , Gu Wei , Oh Tae-Kwang , Kim Myung Hee , Jung Jae U.
Published: April 2009

Cells infected by viruses utilize interferon (IFN)-mediated and p53-mediated irreversible cell cycle arrest and apoptosis as part of the overall host surveillance mechanism to ultimately block viral replication and dissemination. Viruses, in turn, have evolved elaborate mechanisms to subvert IFN- and p53-mediated host innate immune responses. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes several viral IFN regulatory factors (vIRF1 to vIRF4) within a cluster of loci, their functions being primarily to inhibit host IFN-mediated innate immunity and deregulate p53-mediated cell growth control. Despite its significant homology and similar genomic location to other vIRFs, vIRF4 is distinctive, as it does not target and antagonize host IFN-mediated signal transduction. Here, we show that KSHV vIRF4 interacts with the murine double minute 2 (MDM2) E3 ubiquitin ligase, leading to the reduction of p53, a tumor suppressor, via proteasome-mediated degradation. The central region of vIRF4 is required for its interaction with MDM2, which led to the suppression of MDM2 autoubiquitination and, thereby, a dramatic increase in MDM2 stability. Consequently, vIRF4 expression markedly enhanced p53 ubiquitination and degradation, effectively suppressing p53-mediated apoptosis. These results indicate that KSHV vIRF4 targets and stabilizes the MDM2 E3 ubiquitin ligase to facilitate the proteasome-mediated degradation of p53, perhaps to circumvent host growth surveillance and facilitate viral replication in infected cells. Taken together, the indications are that the downregulation of p53-mediated cell growth control is a common characteristic of the four KSHV vIRFs and that p53 is indeed a key factor in the host's immune surveillance program against viral infections.
Vaccinia Virus E3 Suppresses Expression of Diverse Cytokines through Inhibition of the PKR, NF-κB, and IRF3 Pathways
Authors: Myskiw Chad , Arsenio Janilyn , van Bruggen Rebekah , Deschambault Yvon , Cao Jingxin
Published: April 2009

The vaccinia virus double-stranded RNA binding protein E3 has been demonstrated to inhibit the expression of cytokines, including beta interferon (IFN-β) and tumor necrosis factor alpha (TNF-α). However, few details regarding the molecular mechanisms of this inhibition have been described. Using real-time PCR arrays, we found that E3 suppressed the induction of a diverse array of cytokines representing members of the IFN, interleukin (IL), TNF, and transforming growth factor cytokine families. We discovered that the factor(s) responsible for the induction of IL-6, TNF-α, and inhibin beta A (INHBA) was associated with the early and late phases of virus infection. In contrast, the factor(s) which regulates IFN-β induction was associated with the late phase of replication. We have found that expression of these cytokines can be induced by transfection of cells with RNA isolated from vaccinia virus-infected cells. Moreover, we provide evidence that E3 antagonizes both PKR-dependent and PKR-independent pathways to regulate cytokine expression. PKR-dependent activation of p38 and NF-κB was required for vaccinia virus-induced INHBA expression, whereas induction of TNF-α required only PKR-dependent NF-κB activation. In contrast, induction of IL-6 and IFN-β was largely PKR independent. IL-6 induction is regulated by NF-κB, while IFN-β induction is mediated by IFN-β promoter stimulator 1 and IFN regulatory factor 3/NF-κB. Collectively, these results indicate that E3 suppresses distinct but interlinked host signaling pathways to inhibit the expression of a diverse array of cytokines.
Modeling Subgenomic Hepatitis C Virus RNA Kinetics during Treatment with Alpha Interferon
Authors: Dahari Harel , Sainz Bruno , Perelson Alan S. , Uprichard Susan L.
Published: April 2009

Although replicons have been used to demonstrate hepatitis C virus (HCV) inhibition by alpha interferon (IFN-α), the detailed inhibition kinetics required to mathematically model HCV RNA decline have been lacking. Therefore, we measured genotype 1b subgenomic replicon (sg1b) RNA levels under various IFN-α concentrations to assess the inhibition kinetics of intracellular HCV RNA. During nine days of IFN-α treatment, sg1b RNA decreased in a biphasic, dose-dependent manner. Using frequent measurements to dissect these phases during IFN-α treatments of 100 and 250 U/ml revealed that the first-phase sg1b RNA decline began ∼12 h posttreatment, continued for 2 to 4 days, and then exhibited a distinct flat or slower second phase. Based on these data, we developed a mathematical model of IFN-α-induced intracellular sg1b RNA decline, and we show that the mechanism(s) mediating IFN-α inhibition of HCV acts primarily by reducing sg1b RNA amplification, with an additional effect on HCV RNA stability/degradation detectable at a dose of 250 U/ml IFN-α. While the extremely slow or flat second phase of viral RNA inhibition observed in vitro, in which there is little or no cell death, supports the in vivo modeling prediction that the more profound second-phase decline observed in IFN-α-treated patients reflects immune-mediated death/loss of productively infected cells, the second-phase decline in viral RNA with a dose of 250 U/ml IFN-α suggests that a further inhibition of intracellular HCV RNA levels may contribute as well. As such, dissection of HCV IFN-α inhibition kinetics in vitro has brought us closer to understanding the mechanism(s) by which IFN-α may be inhibiting HCV in vivo.
Human Cytomegalovirus Protein pp71 Induces Daxx SUMOylation
Authors: Hwang Jiwon , Kalejta Robert F.
Published: April 2009

Proteins that participate in a diverse array of cellular processes can be modified covalently and reversibly on lysine residues by the small ubiquitin-like modifier proteins termed SUMOs. In some instances, such modification profoundly affects protein function, but the biological significance of many SUMOylation events remains unknown. Protein SUMOylation is modulated during many viral infections. Here we demonstrate that the human cytomegalovirus (HCMV) pp71 protein promotes the SUMOylation of its cellular substrate, Daxx. A component of promyelocytic leukemia nuclear bodies, Daxx is a transcriptional corepressor that silences the expression of viral immediate-early (IE) genes at the start of both lytic and quiescent HCMV infections. pp71 is a tegument component delivered directly to cells by infecting HCMV virions. At the start of lytic infections, it travels to the nucleus and stimulates viral IE gene expression by displacing the chromatin remodeling protein ATRX from Daxx and by mediating Daxx degradation through a rare ubiquitin-independent, proteasome-dependent process. Here we report that pp71 also substantially increases the basal level of SUMOylated Daxx observed in cells. To date, consequences of Daxx SUMOylation have not been observed for cellular promoters, and we detected no qualitative change in viral IE gene expression in the absence of pp71-induced Daxx SUMOylation. Thus, while pp71 enhances the basal level of SUMOylated Daxx, the role that this modification plays in regulating Daxx activity in uninfected or HCMV-infected cells remains an enigma.
ARTHROSCOPIC VISUALISATION OF THE DISTAL RADIOULNAR JOINT
Authors: Yamamoto Michiro , Koh Shukuki , Tatebe Masahiro , Shinohara Takaaki , Shionoya Kaori , Nakamura Ryogo , Hirata Hitoshi
Published: April 2009

The diagnosis of chronic wrist pain is challenging and wrist arthroscopy has been recognised as the "gold standard". The present study investigated the efficacy of adding distal radioulnar joint (DRUJ) arthroscopy to routine wrist arthroscopy. The records of 67 patients who underwent DRUJ arthroscopy were reviewed, and the success rates for visualisation of intra-articular structures were determined. Pathological findings were correlated with ulnar-side wrist pain. In seven patients, pre-operative diagnoses were altered after DRUJ arthroscopy. The ulnar head and proximal surface of the triangular fibrocartilage complex (TFCC) were visualised in 100% and 99% of patients, respectively, while the foveal insertion of TFCC and sigmoid notch were visualised in 57% and 69%, respectively. Pathological findings of the proximal surface of TFCC tended to relate to ulnar wrist pain (p = 0.06). DRUJ arthroscopy should be included in routine wrist arthroscopy to enhance the accuracy of diagnosis.
CLOSED INTRAMEDULLARY FIXATION FOR METACARPAL FRACTURES USING J-SHAPED NAIL
Authors: Itadera Eichi , Hiwatari Ryou , Moriya Hideshige , Ono Yutaka
Published: April 2009

The purpose of this study is to present the results of a new intramedullary fixation technique for metacarpal fractures. A J-shaped nail, which is a curved 2.0 mm diameter Kirschner wire sharply bent at the proximal end, was inserted from the dorsal aspect of the metacarpal base. Twenty-one metacarpal fractures (five transverse shaft fractures and 16 neck fractures) in 19 hands of 18 patients with were operated by this technique. All fractures had successful unions. Clinical and radiographic results were excellent in all ten hands that had excluded concomitant injuries. There was no mechanical irritation of the skin or extensor tendons in any patient. This intramedullary fixation technique is very useful for neck or transverse shaft fractures of the metacarpals without concomitant injuries such as severe soft tissue damage.
A COMPARISON OF ENDOSCOPIC AND OPEN TRIGGER FINGER RELEASE
Authors: Pegoli L. , Cavalli E. , Cortese P. , Parolo C. , Pajardi G.
Published: April 2009

The main complaint of the patients after an open trigger finger release is a discomfort at the incision site. In this prospective study, we compared the two consecutive groups of patients with trigger fingers. One was treated by an open approach and the other by the endoscopic release of the A1 pulley. Pre- and post-operative evaluation at seven, 30 and 90 days showed a faster recovery from the discomfort with a faster return to daily and working activities, after the endoscopic procedure.
EARLY FUNCTIONAL IMPROVEMENT AFTER A MODIFIED LIGAMENT RECONSTRUCTION TENDON INTERPOSITION ARTHROPLASTY FOR THUMB BASAL JOINT ARTHRITIS
Authors: Miura Toshiki , Osuka Koji , Itoh Shozo , Nakagawa Takumi , Kawano Hirotaka , Nakamura Kozo
Published: April 2009

Many modifications to trapeziectomy have been proposed for the treatment of thumb basal joint arthritis. Their final outcomes have been discussed intensively, whereas functional changes in the early post-operative period have not been fully documented. The purpose of the present study is to evaluate an early functional change following our modified ligament reconstruction with tendon interposition (LRTI) arthroplasty. Nine patients (ten thumbs) were included in this study. Pain levels, strength, and mobility were assessed before and after surgery at intervals of two months. Pain level significantly improved at two months after surgery. Grip and pinch strengths had increased gradually after a temporal decrease at two-month follow-up, and were significantly stronger at six months after surgery. Palmar abduction improved significantly at six months after surgery, whereas opposition did not change significantly. A modified LRTI is an effective procedure in terms of early functional improvement of pain, strength, and mobility.
FULL-THICKNESS SKIN GRAFTING IN PSEUDOSYNDACTILY WITH RECESSIVE DYSTROPHIC EPIDERMOLYSIS BULLOSA
Authors: Prasetyono Theddeus O. H. , Yantoko
Published: April 2009

Two 17- and 11-year-old brothers appeared with mitten pseudosyndactily of both hands. They have been suffering from recessive dystrophic epidermolysis bullosa with recurrent cutaneous blister formation either spontaneously or due to minor trauma. Surgeries were performed to release and widen the first web space. Full thickness skin graft which was used to cover the resulting raw surface appears stable as normal skin to them. The two brothers have improvement in range of motion of their thumbs and index fingers.
ANEURYSM OF THE SUPERFICIAL PALMAR ARCH: A CASE REPORT
Authors: Estrella Emmanuel P. , Lee Ellen Y.
Published: April 2009

Aneurysms of the hand are uncommon lesions. The most common location is the ulnar artery. We present a case of a young female who consulted us for a hand mass with a history of trauma to the hand. Pre-operative arteriogram showed a superficial palmar arch aneurysm. The mass was excised and the arch was reconstructed using a reversed Y-shaped vein graft. Fourteen months after surgery, there was no recurrence of the aneurysm and the patient only reported occasional cold intolerance.