bluuebunny/crossref_metadata_2025_split huggingface dataset:
Utilization of the QuantiFERON-TB Gold Test in a Two-Step Process with the Tuberculin Skin Test To Evaluate Health Care Workers for Latent Tuberculosis
Authors: Abdalhamid Baha , Hinrichs Steven H. , Garrett Jodi L. , O'Neill Jean M. , Hansen-Cain Kristine M. , Armbrust Amy A. , Iwen Peter C. 
Published: June 2010

ABSTRACT
A cost analysis of combining a tuberculin skin test (TST) and the QuantiFERON-TB Gold test (QFT-GT) to detect latent tuberculosis in newly hired health care workers was performed. An approximately 50% reduction in the cost of additional care was realized when workers with positive TST results were subsequently screened using the QFT-GT.
Myc Is Required for the Maintenance of Kaposi's Sarcoma-Associated Herpesvirus Latency
Authors: Li Xudong , Chen Shijia , Feng Jun , Deng Hongyu , Sun Ren 
Published: June 2010

ABSTRACT

Myc is deregulated by Kaposi's sarcoma-associated herpesvirus (KSHV) latent proteins, but its role in KSHV latency is not clear. We found that Myc knockdown with RNA interference (RNAi) induced KSHV reactivation and increased the protein and mRNA levels of RTA, a key viral regulator of KSHV reactivation. Myc knockdown increased, whereas Myc overexpression inhibited, RTA promoter activity. KSHV reactivation and the activation of the RTA promoter induced by Myc depletion were inhibited by c-Jun N-terminal kinase (JNK) and p38 inhibitors but not by a MEK1 inhibitor. Myc knockdown inhibited primary effusion lymphoma (PEL) cell proliferation through inducing apoptosis and G
1
cell cycle arrest. Thus, Myc may be a key cellular node coupling cellular transformation and KSHV latency.
Baculovirus GP64 Disulfide Bonds: the Intermolecular Disulfide Bond of
Autographa californica
Multicapsid Nucleopolyhedrovirus GP64 Is Not Essential for Membrane Fusion and Virion Budding
Authors: Li Zhaofei , Blissard Gary W. 
Published: June 2010

ABSTRACT

The GP64 envelope glycoprotein of the
Autographa californica
nucleopolyhedrovirus (AcMNPV) is a class III viral membrane fusion protein that is triggered by low pH during entry. Unlike most other viral fusion protein trimers, the monomers of GP64 are covalently linked to each other within the trimer by a single intermolecular disulfide bond (Cys24—Cys372). Single or paired alanine substitutions for Cys24 and Cys372 resulted in lower-efficiency transport of GP64 to the cell surface. Surprisingly, these mutated GP64s induced syncytium formation, and normalized fusion activities were approximately 30% of that from wild-type (WT) GP64. Heat treatment (37°C) did not further reduce fusion activity of GP64 constructs with a disrupted intermolecular disulfide bond, suggesting that the GP64 trimers were relatively thermostable in the absence of the intermolecular disulfide bond. In addition, analysis of binding by a conformation-specific monoclonal antibody (MAb) suggested that the low-pH-induced refolding of those GP64 constructs was generally similar to that of WT GP64. In addition to its critical role in membrane fusion, GP64 is also necessary for efficient budding. When GP64 constructs containing a disrupted intermolecular disulfide bond (Cys24—Cys372) were displayed at the cell surface at levels comparable to those of WT GP64, virion budding efficiency ranged from approximately 39 to 88%, indicating that the intermolecular disulfide bond is not required for virion budding. However, GP64 proteins with a disrupted intermolecular disulfide could not rescue a GP64-null bacmid. We also examined the 6 conserved intramolecular disulfide bonds using single and paired alanine substitution mutations. None of the GP64 constructs with disrupted intramolecular disulfide bonds were capable of mediating pH-triggered membrane fusion, indicating that the intramolecular disulfide bonds are all necessary for membrane fusion. Thus, while the intramolecular disulfide bonds of GP64 appear to serve critical roles in membrane fusion, the unusual intermolecular disulfide bond was not critical for membrane fusion or virion budding yet appears to play an unknown role in viral infectivity.
Distinct Molecular Pathways to X4 Tropism for a V3-Truncated Human Immunodeficiency Virus Type 1 Lead to Differential Coreceptor Interactions and Sensitivity to a CXCR4 Antagonist
Authors: Del Prete Gregory Q. , Leslie George J. , Haggarty Beth , Jordan Andrea P. O. , Romano Josephine , Hoxie James A. 
Published: June 2010

ABSTRACT
During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed ΔV3-X4A.c5 and ΔV3-X4B.c7, which took distinct molecular pathways for this change. The ΔV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the ΔV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to ΔV3-X4A.c5, ΔV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.
The Cellular RNA Helicase DDX1 Interacts with Coronavirus Nonstructural Protein 14 and Enhances Viral Replication
Authors: Xu Linghui , Khadijah Siti , Fang Shouguo , Wang Li , Tay Felicia P. L. , Liu Ding Xiang 
Published: June 2010

ABSTRACT
The involvement of host proteins in the replication and transcription of viral RNA is a poorly understood area for many RNA viruses. For coronaviruses, it was long speculated that replication of the giant RNA genome and transcription of multiple subgenomic mRNA species by a unique discontinuous transcription mechanism may require host cofactors. To search for such cellular proteins, yeast two-hybrid screening was carried out by using the nonstructural protein 14 (nsp14) from the coronavirus infectious bronchitis virus (IBV) as a bait protein, leading to the identification of DDX1, a cellular RNA helicase in the DExD/H helicase family, as a potential interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation assays with cells coexpressing the two proteins and with IBV-infected cells. Furthermore, the endogenous DDX1 protein was found to be relocated from the nucleus to the cytoplasm in IBV-infected cells. In addition to its interaction with IBV nsp14, DDX1 could also interact with the nsp14 protein from severe acute respiratory syndrome coronavirus (SARS-CoV), suggesting that interaction with DDX1 may be a general feature of coronavirus nsp14. The interacting domains were mapped to the C-terminal region of DDX1 containing motifs V and VI and to the N-terminal portion of nsp14. Manipulation of DDX1 expression, either by small interfering RNA-induced knockdown or by overexpression of a mutant DDX1 protein, confirmed that this interaction may enhance IBV replication. This study reveals that DDX1 contributes to efficient coronavirus replication in cell culture.
Distinct Intracellular Trafficking of Hepatitis C Virus in Myeloid and Plasmacytoid Dendritic Cells
Authors: Lambotin Mélanie , Baumert Thomas F. , Barth Heidi 
Published: June 2010

ABSTRACT

Dendritic cells (DCs) are of pivotal importance for the initiation of immune responses to control and eliminate viral infections. The molecular mechanisms of hepatitis C virus (HCV) antigen uptake and processing by blood DCs are poorly defined. Here we show that human blood DC subsets acquire HCV independent of the classical HCV entry factors. Following HCV uptake, human plasmacytoid and myeloid DC subsets deliver HCV antigen into distinct endocytotic compartments, which are dedicated to presentation to CD4
+
or CD8
+
T cells. Our findings support a model of HCV antigen processing and presentation in which DC subsets fulfill distinct functions.
Mutagenesis of Adeno-Associated Virus Type 2 Capsid Protein VP1 Uncovers New Roles for Basic Amino Acids in Trafficking and Cell-Specific Transduction
Authors: Johnson Jarrod S. , Li Chengwen , DiPrimio Nina , Weinberg Marc S. , McCown Thomas J. , Samulski R. Jude 
Published: June 2010

ABSTRACT

The N termini of the capsid proteins VP1 and VP2 of adeno-associated virus (AAV) play important roles in subcellular steps of infection and contain motifs that are highly homologous to a phospholipase A
2
(PLA
2
) domain and nuclear localization signals (NLSs). To more clearly understand how virion components influence infection, we have generated mutations in these regions and examined their effects on subcellular trafficking, capsid stability, transduction, and sensitivity to pharmacological enhancement. All mutants tested assembled into capsids; retained the correct ratio of VP1, VP2, and VP3; packaged DNA similarly to recombinant AAV2 (rAAV2); and displayed similar stability profiles when heat denatured. Confocal microscopy demonstrated that these mutants trafficked through a perinuclear region in the vicinity of the Golgi apparatus, with a subset of mutants displaying more-diffuse localization consistent with an NLS-deficient phenotype. When tested for viral transduction, two mutant classes emerged. Class I (BR1
−
, BR2
−
, and BR2+K) displayed partial transduction, whereas class II (VP3only,
75
HD/AN, BR3
−
, and BR3+K) were severely defective. Surprisingly, one class II mutant (BR3+K) trafficked identically to rAAV2 and accumulated in the nucleolus, a step recently described by our laboratory that occurs with wild-type infection. The BR3+K mutant, containing an alanine-to-lysine substitution in the third basic region of VP1, was 10- to 100-fold-less infectious than rAAV2 in transformed cell lines (such as HEK-293, HeLa, and CV1-T cells), but in contrast, it was indistinguishable from rAAV2 in several nontransformed cell lines, as well as in tissues (liver, brain, and muscle)
in vivo
. Complementation studies with pharmacological adjuvants or adenovirus coinfection suggested that additional positive charges in NLS regions restrict mobilization in the nucleus and limit transduction in a transformed-cell-specific fashion. Remarkably, besides displaying cell-type-specific transduction, this is the first description of a capsid mutant indicating that nuclear entry is not sufficient for AAV-mediated transduction and suggests that additional steps (i.e., subnuclear mobilization or uncoating) limit successful AAV infection.
Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments
Authors: Nicolas Armel , Alazard-Dany Nathalie , Biollay Coline , Arata Loredana , Jolinon Nelly , Kuhn Lauriane , Ferro Myriam , Weller Sandra K. , Epstein Alberto L. , Salvetti Anna , Greco Anna 
Published: June 2010

ABSTRACT

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV
rep
gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase δ was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.
Identification of Cross-Reactive Norovirus CD4
+
T Cell Epitopes
Authors: LoBue Anna D. , Lindesmith Lisa C. , Baric Ralph S. 
Published: June 2010

ABSTRACT

Immune responses and the components of protective immunity following norovirus infection in humans are poorly understood. Although antibody responses following norovirus infection have been partially characterized, T cell responses in humans remain largely undefined. In contrast, T cells have been shown to be essential for viral clearance of mouse norovirus (MNV) infection. In this paper, we demonstrate that CD4
+
T cells secrete gamma interferon (IFN-γ) in response to stimulation with MNV virus-like particles (VLPs) after MNV infection, supporting earlier reports for norovirus-infected mice and humans. Utilizing this model, we immunized mice with alphavirus vectors (Venezuelan equine encephalitis [VEE] virus replicon particles [VRPs]) expressing Norwalk virus (NV) or Farmington Hills virus (FH) virus-like particles to evaluate T cell epitopes shared between human norovirus strains. Stimulation of splenocytes from norovirus VRP-immunized mice with overlapping peptides from complete libraries of the NV or FH capsid proteins revealed specific amino acid sequences containing T cell epitopes that were conserved within genoclusters and genogroups. Immunization with heterologous norovirus VRPs resulted in specific cross-reactive IFN-γ secretion profiles following stimulation with NV and FH peptides in the mouse. Identification of unique strain-specific and cross-reactive epitopes may provide insight into homologous and heterologous T cell-mediated norovirus immunity and provide a platform for the study of norovirus-induced cellular immunity in humans.
Nucleocytoplasmic Shuttling of Human Cytomegalovirus UL84 Is Essential for Virus Growth
Authors: Gao Yang , Kagele Dominique , Smallenberg Kate , Pari Gregory S. 
Published: June 2010

ABSTRACT
Human cytomegalovirus (HCMV) UL84 is a multifunctional protein that is the proposed initiator for lytic viral DNA synthesis. Recently it was shown that UL84 displays nucleocytoplasmic shuttling. The role of shuttling in lytic DNA replication and virus growth is unknown. We now show that expression of the nonshuttling UL84 mutant failed to complement oriLyt-dependent DNA replication in the transient assay under conditions where core replication and ancillary proteins were expressed under the control of their native promoters. However, constitutive expression of the core replication proteins, along with the nonshuttling UL84 mutant, resulted in efficient oriLyt amplification, suggesting that shuttling may contribute to the activity of one of the auxiliary replication proteins. A recombinant HCMV bacterial artificial chromosome plasmid (BACmid) expressing the nonshuttling UL84 mutant (NS84 BAC) was defective for production of infectious virus. Quantitative PCR showed that NS84 BAC had decreased accumulation of viral DNA in both cellular and supernatant samples. Analysis of the accumulation of select viral mRNAs showed no difference in total cellular mRNA accumulation for IE2, IRS1, TRS1, UL102, UL105, and UL75 in cells transfected with the NS84 BAC. However, examination of cytoplasmic RNA and subcellular localization of IRS1 revealed a decrease in IRS1 mRNA accumulation and displaced protein localization, strongly suggesting that UL84 facilitated the localization of IRS1 mRNA to the cytoplasm. RNA pulldown assays showed that UL84 interacted with IRS1 mRNA. These results indicate that nucleocytoplasmic shuttling is essential for virus growth and strongly suggest that UL84 is responsible for localization of at least one virus-encoded transcript, IRS1 mRNA.
CD25
+
Natural Regulatory T Cells Are Critical in Limiting Innate and Adaptive Immunity and Resolving Disease following Respiratory Syncytial Virus Infection
Authors: Lee Debbie C. P. , Harker James A. E. , Tregoning John S. , Atabani Sowsan F. , Johansson Cecilia , Schwarze Jürgen , Openshaw Peter J. M. 
Published: June 2010

ABSTRACT

Regulatory CD4
+
T cells have been shown to be important in limiting immune responses, but their role in respiratory viral infections has received little attention. Here we observed that following respiratory syncytial virus (RSV) infection, CD4
+
Foxp3
+
CD25
+
natural regulatory T-cell numbers increased in the bronchoalveolar lavage fluid, lung, mediastinal lymph nodes, and spleen. The depletion of CD25
+
natural regulatory T cells prior to RSV infection led to enhanced weight loss with delayed recovery that was surprisingly accompanied by increased numbers of activated natural killer cells in the lung and bronchoalveolar lavage fluid on day 8 postinfection. Increased numbers of neutrophils were also detected within the bronchoalveolar lavage fluid and correlated with elevated levels of myeloperoxidase as well as interleukin-6 (IL-6) and gamma interferon (IFN-γ). CD25
+
natural regulatory T-cell depletion also led to enhanced numbers of proinflammatory T cells producing IFN-γ and tumor necrosis factor alpha (TNF-α) in the lung. Despite these increases in inflammatory responses and disease severity, the viral load was unaltered. This work highlights a critical role for natural regulatory T cells in regulating the adaptive and innate immune responses during the later stages of lung viral infections.
Tyrosines in the Influenza A Virus M2 Protein Cytoplasmic Tail Are Critical for Production of Infectious Virus Particles
Authors: Grantham Michael L. , Stewart Shaun M. , Lalime Erin N. , Pekosz Andrew 
Published: June 2010

ABSTRACT

The cytoplasmic tail of the influenza A virus M2 protein is required for the production of infectious virions. In this study, critical residues in the M2 cytoplasmic tail were identified by single-alanine scanning mutagenesis. The tyrosine residue at position 76, which is conserved in >99% of influenza virus strains sequenced to date, was identified as being critical for the formation of infectious virus particles using both reverse genetics and a protein
trans
-complementation assay. Recombinant viruses encoding M2 with the Y76A mutation demonstrated replication defects in MDCK cells as well as in primary differentiated airway epithelial cell cultures, defects in the formation of filamentous virus particles, and reduced packaging of nucleoprotein into virus particles. These defects could all be overcome by a mutation of serine to tyrosine at position 71 of the M2 cytoplasmic tail, which emerged after blind passage of viruses containing the Y76A mutation. These data confirm and extend our understanding of the significance of the M2 protein for infectious virus particle assembly.
In Vitro
Assembly of the T=13 Procapsid of Bacteriophage T5 with Its Scaffolding Domain
Authors: Huet Alexis , Conway James F. , Letellier Lucienne , Boulanger Pascale 
Published: June 2010

ABSTRACT

The
Siphoviridae
coliphage T5 differs from other members of this family by the size of its genome (121 kbp) and by its large icosahedral capsid (90 nm), which is organized with T=13 geometry. T5 does not encode a separate scaffolding protein, but its head protein, pb8, contains a 159-residue aminoterminal scaffolding domain (Δ domain) that is the mature capsid. We have deciphered the early events of T5 shell assembly starting from purified pb8 with its Δ domain (pb8p). The self assembly of pb8p is regulated by salt conditions and leads to structures with distinct morphologies. Expanded tubes are formed in the presence of NaCl, whereas Ca
2+
promotes the association of pb8p into contracted tubes and procapsids. Procapsids display an angular organization and 20-nm-long internal radial structures identified as the Δ domain. The T5 head maturation protease pb11 specifically cleaves the Δ domain of contracted and expanded tubes. Ca
2+
is not required for proteolytic activity but for the organization of the Δ domain. Taken together, these data indicate that pb8p carries all of the information in its primary sequence to assemble
in vitro
without the requirement of the portal and accessory proteins. Furthermore, Ca
2+
plays a key role in introducing the conformational diversity that permits the formation of a stable procapsid. Phage T5 is the first example of a viral capsid consisting of quasi-equivalent hexamers and pentamers whose assembly can be carried out
in vitro
, starting from the major head protein with its scaffolding domain, and whose endpoint is an icosahedral T=13 particle.
Characterization of the Host Immune Response in Human Ganglia after Herpes Zoster
Authors: Gowrishankar Kavitha , Steain Megan , Cunningham Anthony L. , Rodriguez Michael , Blumbergs Peter , Slobedman Barry , Abendroth Allison 
Published: June 2010

ABSTRACTVaricella-zoster virus (VZV) causes varicella (chicken pox) and establishes latency in ganglia, from where it reactivates to cause herpes zoster (shingles), which is often followed by postherpetic neuralgia (PHN), causing severe neuropathic pain that can last for years after the rash. Despite the major impact of herpes zoster and PHN on quality of life, the nature and kinetics of the virus-immune cell interactions that result in ganglion damage have not been defined. We obtained rare material consisting of seven sensory ganglia from three donors who had suffered from herpes zoster between 1 and 4.5 months before death but who had not died from herpes zoster. We performed immunostaining to investigate the site of VZV infection and to phenotype immune cells in these ganglia. VZV antigen was localized almost exclusively to neurons, and in at least one case it persisted long after resolution of the rash. The large immune infiltrate consisted of noncytolytic CD8+T cells, with lesser numbers of CD4+T cells, B cells, NK cells, and macrophages and no dendritic cells. VZV antigen-positive neurons did not express detectable major histocompatibility complex (MHC) class I, nor did CD8+T cells surround infected neurons, suggesting that mechanisms of immune control may not be dependent on direct contact. This is the first report defining the nature of the immune response in ganglia following herpes zoster and provides evidence for persistence of non-latency-associated viral antigen and inflammation beyond rash resolution.