bluuebunny/crossref_metadata_2025_split huggingface dataset:
RBP-J (CSL) Is Essential for Activation of the K14/vGPCR Promoter of Kaposi's Sarcoma-Associated Herpesvirus by the Lytic Switch Protein RTA
Authors: Liang Yuying , Ganem Don 
Published: June 2004

ABSTRACTThe Kaposi's sarcoma-associated herpesvirus (KSHV) gene product virally encoded G protein-coupled receptor (vGPCR) is a homolog of cellular GPCRs and has been proposed to play important roles in KSHV-induced angiogenesis. The most abundant vGPCR-containing transcripts are K14/vGPCR bicistronic RNAs that are strongly induced during lytic reactivation. Here we show that the promoter governing this transcript is strongly responsive to activation by the viral lytic switch protein RTA. By deletion mapping and scanning mutation analyses, we have identified three putative RTA response elements (A, B, and C) in this promoter. However, none of these sites appear to directly bind RTA in electrophoretic mobility shift assays (EMSA). Site C corresponds to a canonical binding site for RBP-J, a sequence-specific transcriptional repressor that is normally the target of Notch signaling. RBP-J can bind RTA and recruit it to its cognate recognition site; when this happens, the activation function of RTA can relieve RBP-J-mediated repression and upregulate expression of the targeted gene. EMSA studies reveal that both sites A and C can bind to RBP-J; sequence inspection reveals that site A is a novel functional variant of known RBP-J recognition sites. (Site B corresponds to an as-yet-unknown host DNA-binding protein.) The importance of sites A and C in vivo is underscored by the observation that K14/vGPCR promoter function is dramatically inhibited in cells genetically deficient in RBP-J. The regulation of K14/vGPCR transcripts by RBP-J raises the possibility that other modulators of Notch signaling might be able to induce expression of this RNA outside the context of lytic KSHV replication.
Characterization of Infectious Retroviral Pseudotype Particles Bearing Hepatitis C Virus Glycoproteins
Authors: Flint Mike , Logvinoff Carine , Rice Charles M. , McKeating Jane A. 
Published: June 2004

ABSTRACT
The recent development of infectious retroviral pseudotypes bearing hepatitis C virus (HCV) glycoproteins represents an opportunity to study the functionally active form of the HCV E1 and E2 glycoproteins. In the culture supernatant of cells producing HCV retroviral pseudotypes, the majority of E2 was not associated with infectious particles and failed to sediment on sucrose gradients. The E2 that was incorporated into infectious particles appeared as a triplet of diffuse bands at 60, 70, and 90 kDa. Similarly, three major forms of E1 were incorporated into the pseudotype particles, migrating at 33, 31, and 25 kDa. Endoglycosidase H (endo-H) treatment of particles demonstrated that the incorporated E1 was partially or completely sensitive to digestion. In contrast, the majority of the incorporated E2 was endo-H resistant. Purified pseudotype particles were found to contain both disulfide-linked aggregates and nonaggregated E1 and E2. HCV pseudotypes generated from cells expressing E1E2p7 showed similar heterogeneity in the incorporated glycoproteins and were of comparable infectivity to those generated by expression of E1E2. Our results demonstrate the heterogenous nature of E1 and E2 incorporated into retroviral pseudotypes and highlight the difficulty in identifying forms of the HCV glycoproteins that initiate infection.
The Human Immunodeficiency Virus Type 1 Promoter Contains a CATA Box Instead of a TATA Box for Optimal Transcription and Replication
Authors: van Opijnen Tim , Kamoschinski Joost , Jeeninga Rienk E. , Berkhout Ben 
Published: June 2004

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) transcriptional promoter contains a single polymorphism in the TATA box. Most subtypes contain the sequence TATAAGC, but subtype E and some recombinant AG strains have the sequence TA

A

AAGC. Based on mutagenesis studies of cellular RNA polymerase II (pol II) promoters, it has been proposed that the subtype E TATA box is nonfunctional due to the T-to-A substitution at the critical position 3. By means of transcription and virus replication assays, we demonstrate that the true TATA box motif within the viral long terminal repeat (LTR) promoter starts two nucleotides further upstream. Because of this realignment, subtype E has the sequence

CA

TA

A

AA and all other subtypes have the sequence

CA

TATAA. The polymorphism therefore has shifted from position 3 to position 5 and is no longer incompatible with efficient transcription according to rules determined for cellular pol II promoters. In addition, through sensitive competition experiments, we demonstrate that the CATA box of subtypes B and E can be improved for replication by the mutations 1T and 5T, respectively. The fact that the fitness of both subtype LTRs can be increased by specific point mutations in the CATA box suggests that the transcriptional promoter of HIV-1 is fine-tuned towards a suboptimal level of replication. However, this replication rate may be optimal in the in vivo context of an infected individual.
Murine Cytomegalovirus with a Transposon Insertional Mutation at Open Reading Frame m155 Is Deficient in Growth and Virulence in Mice
Authors: Abenes Gerardo , Chan Karen , Lee Manfred , Haghjoo Erik , Zhu Jiaming , Zhou Tianhong , Zhan Xiaoyan , Liu Fenyong 
Published: June 2004

ABSTRACT

A pool of murine cytomegalovirus (MCMV) mutants was previously generated by using a Tn
3
-based transposon mutagenesis approach (X. Zhan, M. Lee, J. Xiao, and F. Liu, J. Virol. 74:7411-7421, 2000). In this study, one of the MCMV mutants, Rvm155, which contained the transposon insertion in open reading frame m155, was characterized in vitro for its replication in tissue culture and in vivo for its growth and virulence in immunodeficient SCID mice. Compared to the wild-type strain and a rescued virus that restored the m155 region, the mutant is significantly deficient in growth in many organs of the infected animals. At 21 days postinfection the titers of Rvm155 in the salivary glands, lungs, spleens, livers, and kidneys of the intraperitoneally infected SCID mice were lower than the titers of the wild-type virus and the rescued virus by 50-, 1,000-, 500-, 100-, and 500-fold, respectively. Moreover, the viral mutant was attenuated in killing the SCID mice, as none of the SCID mice that were intraperitoneally infected with Rvm155 died until 38 days postinfection while all the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results provide the first direct evidence that a disruption of m155 expression leads to attenuation of viral virulence and growth in animals. Moreover, these results suggest that m155 is a viral determinant for optimal MCMV growth and virulence in vivo.
Immunization with Hepatitis C Virus-Like Particles Induces Humoral and Cellular Immune Responses in Nonhuman Primates
Authors: Jeong Sook-Hyang , Qiao Ming , Nascimbeni Michelina , Hu Zongyi , Rehermann Barbara , Murthy Krishna , Liang T. Jake 
Published: June 2004

ABSTRACT

We have previously reported the production of hepatitis C virus-like particles (HCV-LP) using a recombinant baculovirus containing the cDNA of the HCV structural proteins (core, E1, and E2). These particles resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice. Here we present evidence on the immunogenicity of HCV-LP and the effects of novel adjuvant systems in a nonhuman primate model, the baboon. Three groups of four baboons were immunized with HCV-LP, HCV-LP and adjuvant AS01B (monophosphoryl lipid A and QS21), or HCV-LP and the combination of AS01B and CpG oligodeoxynucleotides 10105. After four immunizations over an 8-month period, all animals developed HCV-specific humoral and cellular immune responses including antibodies to HCV structural proteins and gamma interferon
+
(IFN-γ
+
)CD4
+
and IFN-γ
+
CD8
+
T-cell responses. The immunogenicity of HCV-LP was only marginally enhanced by the use of adjuvants. The overall HCV-specific immune responses were broad and long lasting. Our results suggest that HCV-LP is a potent immunogen to induce HCV-specific humoral and cellular immune responses in primates and may be a promising approach to develop novel preventive and therapeutic modalities.
Production of High-Titer Epstein-Barr Virus Recombinants Derived from Akata Cells by Using a Bacterial Artificial Chromosome System
Authors: Kanda Teru , Yajima Misako , Ahsan Nazmul , Tanaka Mika , Takada Kenzo 
Published: June 2004

ABSTRACT

An Epstein-Barr virus (EBV) genome in Burkitt's lymphoma-derived cell line Akata was cloned into a bacterial artificial chromosome (BAC) vector. The BAC clone, designated AK-BAC, was rapidly and precisely modified by means of efficient homologous recombination in
Escherichia coli
. This system was used to produce recombinant EBVs with transgenes. An expression cassette of green fluorescent protein (GFP) was inserted into AK-BAC, and the resultant BAC clone, AK-BAC-GFP, was transfected into Akata cells. We found that transfected BAC plasmids efficiently formed episomes in EBV-positive Akata cells. Mixtures of wild-type and AK-BAC-GFP viruses were then produced and used to infect EBV-negative Akata cells. We obtained cell clones that harbored only AK-BAC-GFP but no wild-type episome. These cell clones produced infectious viruses after stimulating virus production, and the recombinant viruses of AK-BAC-GFP efficiently immortalized primary B lymphocytes. We further revised the method so that any kind of cDNA could be rapidly inserted into the unique I-PpoI site that had been artificially introduced into AK-BAC. The AK-BAC system will have a broad range of applications, such as genetic analyses of various viral gene products and development of viral vectors for human gene therapy.
Sequence Analysis of the Genome of the
Neodiprion sertifer
Nucleopolyhedrovirus
Authors: Garcia-Maruniak Alejandra , Maruniak James E. , Zanotto Paolo M. A. , Doumbouya Aissa E. , Liu Jaw-Ching , Merritt Thomas M. , Lanoie Jennifer S. 
Published: June 2004

ABSTRACT

The genome of the
Neodiprion sertifer
nucleopolyhedrovirus (NeseNPV), which infects the European pine sawfly,
N. sertifer
(Hymenoptera: Diprionidae), was sequenced and analyzed. The genome was 86,462 bp in size. The C+G content of 34% was lower than that of the majority of baculoviruses. A total of 90 methionine-initiated open reading frames (ORFs) with more than 50 amino acids and minimal overlapping were found. From those, 43 ORFs were homologous to other baculovirus ORFs, and 29 of these were from the 30 conserved core genes among all baculoviruses. A NeseNPV homolog to the
ld130
gene, which is present in all other baculovirus genomes sequenced to date, could not be identified. Six NeseNPV ORFs were similar to non-baculovirus-related genes, one of which was a trypsin-like gene. Only one
iap
gene, containing a single BIR motif and a RING finger, was found in NeseNPV. Two NeseNPV ORFs (nese18 and nese19) were duplicates transcribed in opposite orientations from each other. NeseNPV did not have an AcMNPV ORF 2 homolog characterized as the baculovirus repeat ORF (
bro
). Six homologous regions (
hr
s) were located within the NeseNPV genome, each containing small palindromes embedded within direct repeats. A phylogenetic analysis was done to root the tree based upon the sequences of DNA polymerase genes of NeseNPV, 23 other baculoviruses, and other phyla. Baculovirus phylogeny was then constructed with 29 conserved genes from 24 baculovirus genomes.
Culex nigripalpus
nucleopolyhedrovirus (CuniNPV) was the most distantly related baculovirus, branching to the hymenopteran NeseNPV and the lepidopteran nucleopolyhedroviruses and granuloviruses.
Protection against Lethal Vaccinia Virus Challenge in HLA-A2 Transgenic Mice by Immunization with a Single CD8+T-Cell Peptide Epitope of Vaccinia and Variola Viruses
Authors: Snyder James T. , Belyakov Igor M. , Dzutsev Amiran , Lemonnier François , Berzofsky Jay A. 
Published: June 2004

ABSTRACTCD8+T lymphocytes have been shown to be involved in controlling poxvirus infection, but no protective cytotoxic T-lymphocyte (CTL) epitopes are defined for variola virus, the causative agent of smallpox, or for vaccinia virus. Of several peptides in vaccinia virus predicted to bind HLA-A2.1, three, VETFsm(498-506), A26L(6-14), and HRP2(74-82), were found to bind HLA-A2.1. Splenocytes from HLA-A2.1 transgenic mice immunized with vaccinia virus responded only to HRP2(74-82) at 1 week and to all three epitopes by ex vivo enzyme-linked immunosorbent spot (ELISPOT) assay at 4 weeks postimmunization. To determine if these epitopes could elicit a protective CD8+T-cell response, we challenged peptide-immunized HLA-A2.1 transgenic mice intranasally with a lethal dose of the WR strain of vaccinia virus. HRP2(74-82) peptide-immunized mice recovered from infection, while naïve mice died. Depletion of CD8+T cells eliminated protection. Protection of HHD-2 mice, lacking mouse class I major histocompatibility complex molecules, implicates CTLs restricted by human HLA-A2.1 as mediators of protection. These results suggest that HRP2(74-82), which is shared between vaccinia and variola viruses, may be a CD8+T-cell epitope of vaccinia virus that will provide cross-protection against smallpox in HLA-A2.1-positive individuals, representing almost half the population.
Subunit-Specific Analysis of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase In Vivo
Authors: Mulky Alok , Sarafianos Stefan G. , Arnold Edward , Wu Xiaoyun , Kappes John C. 
Published: June 2004

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer comprised of two structurally distinct subunits (p51 and p66). Since p51 and p66 are derived from the same coding region, subunit-specific structure-function studies of RT have been conducted exclusively by in vitro biochemical approaches. To study RT subunit function in the context of infectious virus, we constructed an LTR-vpr-p51-IRES-p66 expression cassette in which the HIV-1
vpr
gene was fused in frame with p51, followed by an internal ribosome entry site (IRES) sequence and the p66 coding region. By coexpression with RT-deficient proviral DNA, we demonstrated that the p66 subunit is specifically and selectively packaged into virions as a Vpr-p51/p66 complex. Our analysis showed that cleavage by the viral protease liberates Vpr and generates functional heterodimeric RT (p51/p66) that supports HIV-1 reverse transcription and virus infection. By exploiting this novel
trans
-complementation approach, we demonstrated, for the first time with infectious virions, that the YMDD aspartates of p66 are both required and sufficient for RT polymerase function. Mutational analyses of the p51 YMDD aspartates indicated that they play an important structural role in p51 folding and subunit interactions that are required for the formation of an active RT heterodimer within infected cells. Understanding the role of the individual RT subunits in RNA- and DNA-dependent DNA synthesis is integral to our understanding of RT function. Our findings will lead to important new insights into the role of the p51 and p66 subunits in HIV-1 reverse transcription.
Human Monoclonal Antibody to Hepatitis C Virus E1 Glycoprotein That Blocks Virus Attachment and Viral Infectivity
Authors: Keck Zhen-Yong , Sung Vicky M. H. , Perkins Susan , Rowe Judy , Paul Sudhir , Liang T. Jake , Lai Michael M. C. , Foung Steven K. H. 
Published: June 2004

ABSTRACT
Human antibodies elicited in response to hepatitis C virus (HCV) infection are anticipated to react with the native conformation of the viral envelope structure. Isolation of these antibodies as human monoclonal antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. H-111, an antibody to HCV envelope 1 protein (E1) that maps to the YEVRNVSGVYH sequence and is located near the N terminus of E1 and is able to immunoprecipitate E1E2 heterodimers, is described. Binding of H-111 to HCV E1 genotypes 1a, 1b, 2b, and 3a indicates that the H-111 epitope is highly conserved. Sequence analysis of antibody V regions showed evidence of somatic and affinity maturation of H-111. Finally, H-111 blocks HCV-like particle binding to and HCV virion infection of target cells, suggesting the involvement of this epitope in virus binding and entry.
Human Immunodeficiency Virus Type 1 (HIV-1) Diversity at Time of Infection Is Not Restricted to Certain Risk Groups or Specific HIV-1 Subtypes
Authors: Sagar Manish , Kirkegaard Erin , Long E. Michelle , Celum Connie , Buchbinder Susan , Daar Eric S. , Overbaugh Julie 
Published: June 2004

ABSTRACT
African women frequently acquire several genetically distinct human immunodeficiency virus type 1 (HIV-1) variants from a heterosexual partner, whereas the acquisition of multiple variants appears to be rare in men. To determine whether newly infected individuals in other risk groups acquire genetically diverse viruses, we examined the viral envelope sequences in plasma samples from 13 women and 4 men from the United States infected with subtype B viruses and 10 men from Kenya infected with non-subtype B viruses. HIV-1 envelope sequences differed by more than 2% in three U.S. women, one U.S. man, and one Kenyan man near the time of seroconversion. These findings suggest that early HIV-1 genetic diversity is not exclusive to women from Africa or to infection with any particular HIV-1 subtype.
Ex Vivo Phenotype and Frequency of Influenza Virus-Specific CD4 Memory T Cells
Authors: Lucas Michaela , Day Cheryl L. , Wyer Jessica R. , Cunliffe Sharon L. , Loughry Andrew , McMichael Andrew J. , Klenerman Paul 
Published: June 2004

ABSTRACTRecent advances in class II tetramer staining technology have allowed reliable direct ex vivo visualization of antigen-specific CD4 T cells. In order to define the frequency and phenotype of a prototype response to a nonpersistent pathogen, we have used such techniques to analyze influenza virus-specific memory CD4 T cells directly from blood. These responses are stably detectable ex vivo at low frequencies (range, 0.00012 to 0.0061% of CD4 T cells) and display a distinct “central memory” CD62L+phenotype.
Procalcitonin and C-reactive protein during systemic inflammatory response syndrome, sepsis and organ dysfunction
Authors: Castelli Gian Paolo , Pognani Claudio , Meisner Michael , Stuani Antonio , Bellomi Daniela , Sgarbi Laura 
Published: June 2004

Abstract
Introduction
Both C-reactive protein (CRP) and procalcitonin (PCT) are accepted sepsis markers. However, there is still some debate concerning the correlation between their serum concentrations and sepsis severity. We hypothesised that PCT and CRP concentrations are different in patients with infection or with no infection at a similar severity of organ dysfunction or of systemic inflammatory response.

Patients and methods
One hundred and fifty adult intensive care unit patients were observed consecutively over a period of 10 days. PCT, CRP and infection parameters were compared among the following groups: no systemic inflammatory response syndrome (SIRS) (n = 15), SIRS (n = 15), sepsis/SS (n = 71) (including sepsis, severe sepsis and septic shock [n = 34, n = 22 and n = 15]), and trauma patients (n = 49, no infection).

Results
PCT and CRP concentrations were higher in patients in whom infection was diagnosed at comparable levels of organ dysfunction (infected patients, regression of median [ng/ml] PCT = -0.848 + 1.526 sequential organ failure assessment [SOFA] score, median [mg/l] CRP = 105.58 + 0.72 SOFA score; non-infected patients, PCT = 0.27 + 0.02 SOFA score, P < 0.0001; CRP = 84.53 - 0.19 SOFA score, P < 0.005), although correlation with the SOFA score was weak (R = 0.254, P < 0.001 for PCT, and R = 0.292, P < 0.001 for CRP). CRP levels were near their maximum already during lower SOFA scores, whereas maximum PCT concentrations were found at higher score levels (SOFA score > 12).
PCT and CRP concentrations were 1.58 ng/ml and 150 mg/l in patients with sepsis, 0.38 ng/ml and 51 mg/l in the SIRS patients (P < 0.05, Mann–Whitney U-test), and 0.14 ng/ml and 72 mg/l in the patients with no SIRS (P < 0.05). The kinetics of both parameters were also different, and PCT concentrations reacted more quickly than CRP.

Conclusions
PCT and CRP levels are related to the severity of organ dysfunction, but concentrations are still higher during infection. Different sensitivities and kinetics indicate a different clinical use for both parameters.
Active Transport of Nitrofurantoin into Human Milk
Authors: Gerk Phillip M. , Kuhn Robert J. , Desai Nirmala S. , McNamara Patrick J. 
Published: June 2004

Study Objective. To determine the extent to which nitrofurantoin is transferred into human milk.Design. Prospective, single‐dose pharmacokinetic study.Setting. University‐affiliated clinical research center.Patients. Four healthy lactating women 8–26 weeks postpartum.Intervention. All subjects received a single, oral, 100‐mg dose of nitrofurantoin macrocrystals with food. Serial serum and milk samples were obtained and analyzed by high‐performance liquid chromatography.Measurements and Main Results. Milk pH, milk fat partitioning, and protein binding in serum and milk were determined. Predicted milk:serum ratio (M:S) was compared with the observed M:S. Nitrofurantoin M:S predicted was 0.28 ± 0.05, whereas M:S observed was 6.21 ± 2.71. Average milk concentration was 1.3 mg/L, and estimated suckling infant dosage was 0.2 mg/kg/day or 6% of maternal dose (mg/kg).Conclusions. Nitrofurantoin is actively transported into human milk, achieving concentrations in milk greatly exceeding those in serum. Concern is warranted for suckling infants younger than 1 month old, or for infants with a high frequency of glucose‐6–phosphate dehydrogenase deficiency or sensitivity to nitrofurantoin.
Economic Impact of Neutropenia and Febrile Neutropenia in Breast Cancer: Estimates from Two National Databases
Authors: Gandhi Sanjay K. , Arguelles Lester , Boyer J. Gregory 
Published: June 2004

Study Objective. To estimate the economic burden of neutropenia and febrile neutropenia in female breast cancer hospital admissions in the United States.Design. Retrospective database analysis.Patients. Female admissions with a breast cancer diagnosis.Measurements and Main Results. By reviewing two national databases (Healthcare Costs and Utilization Project, MarketScan), length of stay and charge or payment/admission were estimated from 1994–1996. Neutropenic and febrile neutropenic admissions were longer and incurred higher charges and payments than nonneutropenic and afebrile neutropenic admissions, respectively (p<0.05). The difference in mean charges between neutropenic and nonneutropenic admissions decreased from $13,143 in 1994 to $6913 in 1996, whereas the difference in payment was $4957 (adjusted to 1996 dollars). The difference in mean charges between febrile and afebrile neutropenic admissions decreased from $11,570 in 1994 to $2873 in 1996, whereas the difference in payment was $2390 (adjusted to 1996 dollars).Conclusion. There was a trend toward decreased charges for inpatient admissions with neutropenia in patients with breast cancer (1994–1996). Interventions that reduce the frequency of neutropenia and febrile neutropenia could reduce hospitalization costs of breast cancer admissions.