bluuebunny/crossref_metadata_2025 huggingface dataset:
Posthospital Care Transitions: Patterns, Complications, and Risk Identification
Authors: Coleman Eric A. , Min Sung‐joon , Chomiak Alyssa , Kramer Andrew M.
Published: August 2004

Objectives. To (1) describe patterns of posthospital care transitions; (2) characterize these patterns as uncomplicated or complicated; (3) identify those at greatest risk for complicated transitions.

Data Sources/Study Setting. The Medicare Current Beneficiary Survey was used to identify beneficiaries aged 65 and older who were discharged from an acute care hospital in 1997–1998.

Study Design. Patterns of posthospital transfers were described over a 30‐day time period following initial hospital discharge. Uncomplicated posthospital care patterns were defined as a sequence of transfers from higher‐to lower‐intensity care environments without recidivism, while complicated posthospital care patterns were defined as the opposite sequence of events. Indices were developed to identify patients at risk for complicated transitions.

Principal Findings. Forty‐six distinct types of care patterns were observed during the 30 days following hospital discharge. Among these patterns, 444 episodes (61.2 percent) were limited to a single transfer, 130 episodes (17.9 percent) included two transfers, 62 episodes (8.5 percent) involved three transfers, and 31 episodes (4.3 percent) involved four or more transfers. Fifty‐nine episodes (8.1 percent) resulted in death. Between 13.4 percent and 25.0 percent of posthospital care patterns in the 1998 sample were classified as complicated. The area under the receiver operating curve was 0.771 for a predictive index that utilized administrative data and 0.833 for an index that used a combination of administrative and self‐reported data.

Conclusions. Posthospital care transitions are common among Medicare beneficiaries and patterns of care vary greatly. A significant number of beneficiaries experienced complicated care transitions—a finding that has important implications for both patient safety and cost‐containment efforts. Patients at risk for complicated care patterns can be identified using data available at the time of hospital discharge.
Plan, Geographical, and Temporal Variation of Consumer Assessments of Ambulatory Health Care
Authors: Zaslavsky Alan M. , Zaborski Lawrence B. , Cleary Paul D.
Published: August 2004

Objective. To quantify contributions of health plans and geography to variation in consumer assessments of health plan quality.

Data Sources. Responses of beneficiaries of Medicare managed care plans to the Consumer Assessment of Health Plans Study (CAHPS®) survey. Our data included more than 700,000 survey responses assessing 381 Medicare managed care (MMC) contracts over a period of five years.

Study Design. The survey was administered to a nationally representative sample of beneficiaries of Medicare managed care plans.

Principal Findings. Member assessments of their health plans, customer service functions, and prescription drug benefits varied most across health plans; these also varied the most over time. Assessments of direct interactions with doctors and their practices were more affected by geographical location, and these assessments were quite stable over time. A health plan's global rating often changed significantly between consecutive years, but only rarely were there such changes in ratings of care or doctor. Nationally, mean assessments tended to decrease over the study period.

Conclusions. Our findings suggest that ratings of plans and reports about customer service and prescription access are affected by plan policies, benefits design, and administrative structures that can be changed relatively quickly. Conversely, assessments of other aspects of care are largely determined by characteristics of provider networks that are relatively stable. A consumer survey is unlikely to detect meaningful changes in quality of care from year to year unless quality improvement measures are developed that have substantially larger effects, possibly through area‐wide initiatives, than historical temporal variations in quality.
The Unintended Impact of Welfare Reform on the Medicaid Enrollment of Eligible Immigrants
Authors: Kandula Namratha R. , Grogan Colleen M. , Rathouz Paul J. , Lauderdale Diane S.
Published: August 2004

Background. During welfare reform, Congress passed legislation barring legal immigrants who entered the United States after August 1996 from Medicaid for five years after immigration. This legislation intended to bar only new immigrants (post‐1996 immigrants) from Medicaid. However it may have also deterred the enrollment of legal immigrants who immigrated before 1996 (pre‐1996 immigrants) and who should have remained Medicaid eligible.

Objectives. To compare the Medicaid enrollment of U.S.‐born citizens to pre‐1996 immigrants, before and after welfare reform, and to determine if variation in state Medicaid policies toward post‐1996 immigrants modified the effects of welfare reform on pre‐1996 immigrants.

Data Source/Study Design. Secondary database analysis of cross‐sectional data from 1994–2001 of the U.S. Census Bureau, Annual Demographic Survey of March Supplement of the Current Population Survey.

Subjects. Low‐income, U.S.‐born adults (N=116,307) and low‐income pre‐1996 immigrants (N=24,367) before and after welfare reform.

Measures. Self‐reported Medicaid enrollment.

Results. Before welfare reform, pre‐1996 immigrants were less likely to enroll in Medicaid than the U.S.‐born (OR=0.55; 95 percent CI, 0.51–0.59). After welfare reform, pre‐1996 immigrants were even less likely to enroll in Medicaid. The proportion of immigrants in Medicaid dropped 3 percentage points after 1996; for the U.S.‐born it dropped 1.6 percentage points (p=0.012). Except for California, state variation in Medicaid policy toward post‐1996 immigrants did modify the effect of welfare reform on pre‐1996 immigrants.

Conclusions. Federal laws limiting the Medicaid eligibility of specific subgroups of immigrants appear to have had unintended consequences on Medicaid enrollment in the larger, still eligible immigrant community. Inclusive state policies may overcome this effect.
Subsidies and the Demand for Individual Health Insurance in California
Authors: Susan Marquis M. , Buntin Melinda Beeuwkes , Escarce José J. , Kapur Kanika , Yegian Jill M.
Published: August 2004

Objective. To estimate the effect of changes in premiums for individual insurance on decisions to purchase individual insurance and how this price response varies among subgroups of the population.

Data Source. Survey responses from the Current Population Survey (http://www.bls.census.gov/cps/cpsmain.htm), the Survey of Income and Program Participation (http://www.sipp.census.gov/sipp), the National Health Interview Survey (http://www.cdc.gov/nchs/nhis.htm), and data about premiums and plans offered in the individual insurance market in California, 1996–2001.

Study Design. A logit model was used to estimate the decisions to purchase individual insurance by families without access to group insurance. This was modeled as a function of premiums, controlling for family characteristics and other characteristics of the market. A multinomial model was used to estimate the choice between group coverage, individual coverage, and remaining uninsured for workers offered group coverage as a function of premiums for individual insurance and out‐of‐pocket costs of group coverage.

Principal Findings. The elasticity of demand for individual insurance by those without access to group insurance is about −.2 to −.4, as has been found in earlier studies. However, there are substantial differences in price responses among subgroups with low‐income, young, and self‐employed families showing the greatest response. Among workers offered group insurance, a decrease in individual premiums has very small effects on the choice to purchase individual coverage versus group coverage.

Conclusions. Subsidy programs may make insurance more affordable for some families, but even sizeable subsidies are unlikely to solve the problem of the uninsured. We do not find evidence that subsidies to individual insurance will produce an unraveling of the employer‐based health insurance system.
E Protein Silencing by the Leukemogenic AML1-ETO Fusion Protein
Authors: Zhang Jinsong , Kalkum Markus , Yamamura Soichiro , Chait Brian T. , Roeder Robert G.
Published: August 2004

The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 15% of acute myeloid leukemia (AML) cases. This study shows that AML1-ETO, as well as ETO, inhibits transcriptional activation by E proteins through stable interactions that preclude recruitment of p300/CREB-binding protein (CBP) coactivators. These interactions are mediated by a conserved ETO TAF4 homology domain and a 17–amino acid p300/CBP and ETO target motif within AD1 activation domains of E proteins. In t(8;21) leukemic cells, very stable interactions between AML1-ETO and E proteins underlie a t(8;21) translocation-specific silencing of E protein function through an aberrant cofactor exchange mechanism. These studies identify E proteins as AML1-ETO targets whose dysregulation may be important for t(8;21) leukemogenesis, as well as an E protein silencing mechanism that is distinct from that associated with differentiation-inhibitory proteins.
Chiral-Selective Aminoacylation of an RNA Minihelix
Authors: Tamura Koji , Schimmel Paul
Published: August 2004

Amino acids in natural proteins have a chiral, asymmetric center at the α carbon that is of the
L
-configuration. The sugar backbone of natural RNAs are also homochiral, but of the
D
-configuration. Because protein synthesis requires the aminoacylation of RNA, it is this step that could have provided chiral selectivity. Here we show that an RNA minihelix was aminoacylated by an aminoacyl-phosphate-
D
-oligonucleotide with a clear preference for
L
- as opposed to
D
-amino acids. A mirror-image RNA system showed the opposite selectivity. These results suggest the possibility that the selection of
L
-amino acids for proteins was determined by the stereochemistry of RNA.
Nanoribbon Waveguides for Subwavelength Photonics Integration
Authors: Law Matt , Sirbuly Donald J. , Johnson Justin C. , Goldberger Josh , Saykally Richard J. , Yang Peidong
Published: August 2004

Although the electrical integration of chemically synthesized nanowires has been achieved with lithography, optical integration, which promises high speeds and greater device versatility, remains unexplored. We describe the properties and functions of individual crystalline oxide nanoribbons that act as subwavelength optical waveguides and assess their applicability as nanoscale photonic elements. The length, flexibility, and strength of these structures enable their manipulation on surfaces, including the optical linking of nanoribbon waveguides and other nanowire elements to form networks and device components. We demonstrate the assembly of ribbon waveguides with nanowire light sources and detectors as a first step toward building nanowire photonic circuitry.
Impaired Degradation of Mutant α-Synuclein by Chaperone-Mediated Autophagy
Authors: Cuervo Ana Maria , Stefanis Leonidas , Fredenburg Ross , Lansbury Peter T. , Sulzer David
Published: August 2004

Aberrant α-synuclein degradation is implicated in Parkinson's disease pathogenesis because the protein accumulates in the Lewy inclusion bodies associated with the disease. Little is known, however, about the pathways by which wild-type α-synuclein is normally degraded. We found that wild-type α-synuclein was selectively translocated into lysosomes for degradation by the chaperone-mediated autophagy pathway. The pathogenic A53T and A30P α-synuclein mutants bound to the receptor for this pathway on the lysosomal membrane, but appeared to act as uptake blockers, inhibiting both their own degradation and that of other substrates. These findings may underlie the toxic gain-of-function by the mutants.
Genome Size and Structure Determine Efficiency of Postinternalization Steps and Gene Transfer of Capsid-Modified Adenovirus Vectors in a Cell-Type-Specific Manner
Authors: Shayakhmetov Dmitry M. , Li Zong-Yi , Gaggar Anuj , Gharwan Helen , Ternovoi Vladimir , Sandig Volker , Lieber André
Published: August 2004

Adenovirus serotype 5 (Ad5) vectors containing Ad B-group fibers have become increasingly popular as gene transfer vectors because they efficiently transduce human cell types that are relatively refractory to Ad5 infection. So far, most B-group fiber-containing vectors have been first-generation vectors, deleted of E1 and/or E3 genes. Transduction with these vectors, however, results in viral gene expression and is associated with cytotoxicity and immune responses against transduced cells. To circumvent these problems, we developed fiber-chimeric Ad vectors devoid of all viral genes that were produced either by the homologous recombination of first-generation vectors or by using the Cre/lox-based helper virus system. In this study we compared early steps of infection between first-generation (35-kb genome) and Ad vectors devoid of all viral genes with genome sizes of 28 kb and 12.6 kb. All vectors possessed an Ad35-derived fiber knob domain, which uses CD46 as a primary attachment receptor. Using immortalized human hematopoietic cell lines and primary human CD34-positive hematopoietic cells, we found that the Ad genome size did not affect the efficiency of virus attachment to and internalization into cells. Furthermore, independently of the genome length and structure, all vectors migrated to the nucleus through late endosomal and lysosomal cellular compartments. However, the vector containing the short 12.6-kb genome was unable to efficiently escape from endosomes and deliver its DNA into the nucleus. Moreover, compared to other vectors, these Ad particles were less stable and had an abnormal capsid protein composition, including a lack of capsid-stabilizing protein IX. Our data indicate that the size and structure of the packaged viral genomes can affect the integrity of Ad particles, which in turn results in lower infectivity of Ad vectors.
Human Cytomegalovirus UL131-128 Genes Are Indispensable for Virus Growth in Endothelial Cells and Virus Transfer to Leukocytes
Authors: Hahn Gabriele , Revello Maria Grazia , Patrone Marco , Percivalle Elena , Campanini Giulia , Sarasini Antonella , Wagner Markus , Gallina Andrea , Milanesi Gabriele , Koszinowski Ulrich , Baldanti Fausto , Gerna Giuseppe
Published: August 2004

Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects and morbidity in immunocompromised patients and a potential trigger for vascular disease. HCMV replicates in vascular endothelial cells and drives leukocyte-mediated viral dissemination through close endothelium- leukocyte interaction. However, the genetic basis of HCMV growth in endothelial cells and transfer to leukocytes is unknown. We show here that the UL131-128 gene locus of HCMV is indispensable for both productive infection of endothelial cells and transmission to leukocytes. The experimental evidence for this is based on both the loss-of-function phenotype in knockout mutants and natural variants and the gain-of-function phenotype by
trans
-complementation with individual UL131, UL130, and UL128 genes. Our findings suggest that a common mechanism of virus transfer may be involved in both endothelial cell tropism and leukocyte transfer and shed light on a crucial step in the pathogenesis of HCMV infection.
Transcriptional Activation by the Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Is Facilitated by an N-Terminal Chromatin-Binding Motif
Authors: Wong Lai-Yee , Matchett Gerald A. , Wilson Angus C.
Published: August 2004

In immunocompromised patients, infection with Kaposi's sarcoma-associated herpesvirus (KSHV) can give rise to Kaposi's sarcoma and several lymphoproliferative disorders. In these tumors, KSHV establishes a latent infection in many of the rapidly proliferating and morphologically abnormal cells. Only a few viral gene products are expressed by the latent virus, and one of the best characterized is the latency-associated nuclear antigen (LANA), a nuclear protein required for the maintenance of viral episomal DNA in the dividing host cell. LANA can also activate or repress an assortment of cellular and viral promoters and may contribute to pathogenesis by allowing the proliferation and survival of host cells. Here we show that activation of the human E2F1 and cyclin-dependent kinase-2 (CDK2) promoters requires elements from both the N- and C-terminal regions of LANA. Deletion of the first 22 amino acids, which are necessary for episome tethering, does not affect nuclear localization but significantly reduces transactivation. Within the deleted peptide, we have identified a short sequence, termed the chromatin-binding motif (CBM), that binds tightly to interphase and mitotic chromatin. A second chromatin-binding activity resides in the C terminus but is not sufficient for optimal transactivation. Alanine substitutions within the CBM reveal a close correlation between the transactivation and chromatin binding activities, implying a mechanistic link. In contrast to promoter activation, we find that the 223 amino acids of the LANA C terminus are sufficient to inhibit p53-mediated activation of the human BAX promoter, indicating that the CBM is not required for all transcription-related functions.
Re-Evaluating Natural Resistance to Herpes Simplex Virus Type 1
Authors: Halford William P. , Balliet John W. , Gebhardt Bryan M.
Published: August 2004

It is often stated that individuals of a species can differ significantly in their innate resistance to infection with herpes simplex virus type 1 (HSV-1). Three decades ago Lopez reported that C57BL/6 mice could survive a 5,000-fold-higher inoculum of HSV-1 given intraperitoneally than mice of the A or BALB/c strain (Nature258:152-153, 1975). Susceptible strains of mice died of encephalitis-like symptoms, suggesting that viral spread to the central nervous system was the cause of death. Although Lopez's study documented that C57BL/6 mice were resistant to the development of HSV-1 encephalitis and mortality, the resistance of C57BL/6 mice to other steps of the HSV-1 infection process was not assessed. The results of the present study extend these observations to clarify the difference between resistance to (i) HSV-1 pathogenesis, (ii) HSV-1 replication, (iii) HSV-1 spread, and (iv) the establishment of latent HSV-1 infection. Although C57BL/6 mice are more resistant to HSV-1 pathogenesis than BALB/c mice, the results of the present study establish that HSV-1 enters, replicates, spreads, and establishes latent infections with virtually identical efficiencies in C57BL/6 and BALB/c mice. These observations raise questions about the validity of the inference that differences in natural resistance are relevant in explaining what differentiates humans with recurrent herpetic disease from the vast majority of asymptomatic carriers of HSV-1 and HSV-2.
Kinetics of Virus-Specific CD8+T Cells and the Control of Human Immunodeficiency Virus Infection
Authors: Davenport Miles P. , Ribeiro Ruy M. , Perelson Alan S.
Published: August 2004

Several primate models indicate that cytotoxic T lymphocyte-inducing vaccines may be unable to prevent human immunodeficiency virus infection but may have a long-term benefit in controlling viral replication and delaying disease progression. Here we show that analysis of the kinetics of antigen-specific CD8+T-cell expansion suggests a delay in activation following infection that allows unimpeded early viral replication. Viral kinetics do not differ between controls and vaccinees during this delay phase. An increase in virus-specific CD8+T-cell numbers around day 10 postinfection coincides with a slowing in viral replication in vaccinees and reduces peak viral loads by around 1 log. However, this response is too little too late to prevent establishment of persistent infection.
Helper-Dependent Adenoviral Vector-Mediated Delivery of Woodchuck-Specific Genes for Alpha Interferon (IFN-α) and IFN-γ: IFN-α but Not IFN-γ Reduces Woodchuck Hepatitis Virus Replication in Chronic Infection In Vivo
Authors: Fiedler Melanie , Rödicker Florian , Salucci Valentina , Lu Mengji , Aurisicchio Luigi , Dahmen Uta , Jun Li , Dirsch Olaf , Pützer Brigitte M. , Palombo Fabio , Roggendorf Michael
Published: August 2004

Alpha interferon (IFN-α) and IFN-γ are able to suppress hepadnavirus replication. The intrahepatic expression of high levels of IFN may enhance the antiviral activity. We investigated the effects of woodchuck-specific IFN-α (wIFN-α) and IFN-γ(wIFN-γ) on woodchuck hepatitis virus (WHV) replication in vivo by helper-dependent adenoviral (HD-Ad) vector-mediated gene transfer. The expression of biologically active IFNs was demonstrated in vitro after transduction of woodchuck cells with HD-Ad vectors encoding wIFN-α (HD-AdwIFN-α) or wIFN-γ (HD-AdwIFN-γ). The transduction efficacy of the HD-Ad vector in woodchuck liver in vivo was tested with a vector expressing green fluorescence protein (GFP). Immunohistochemical staining of liver samples on day 5 after injection showed expression of GFP in a high percentage of liver cells surrounding the central vein. The transduction of livers of WHV carriers in vivo with HD-AdwIFN-α or HD-AdwIFN-γ induced levels of biologically active IFN, which could be measured in the sera of these animals. Expression of wIFN-α in the liver reduced intrahepatic WHV replication and WHV DNA in sera of about 1 log step in two of two woodchucks. Transduction with HD-AdwIFN-γ, however, reduced WHV replicative intermediates only slightly in two of three animals, which was not accompanied with significant changes in the WHV DNA in sera. We demonstrated for the first time the successful HD-Ad vector-mediated transfer of genes for IFN-α and IFN-γ in vivo and timely limited reduction of WHV replication by wIFN-α, but not by wIFN-γ.
Bidirectional Increase in Permeability of Nuclear Envelope upon Poliovirus Infection and Accompanying Alterations of Nuclear Pores
Authors: Belov George A. , Lidsky Peter V. , Mikitas Olga V. , Egger Denise , Lukyanov Konstantin A. , Bienz Kurt , Agol Vadim I.
Published: August 2004

Poliovirus and some other picornaviruses trigger relocation of certain nuclear proteins into the cytoplasm. Here, by using a protein changing its fluorescence color with time and containing a nuclear localization signal (NLS), we demonstrate that the poliovirus-triggered relocation is largely due to the exit of presynthesized nuclear protein into the cytoplasm. The leakiness of the nuclear envelope was also documented by the inability of nuclei from digitonin-permeabilized, virus-infected (but not mock-infected) cells to retain an NLS-containing derivative of green fluorescent protein (GFP). The cytoplasm-to-nucleus traffic was also facilitated during infection, as evidenced by experiments with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), cyclin B1, and an NLS-lacking derivative of GFP, which are predominantly cytoplasmic in uninfected cells. Electron microscopy demonstrated that a bar-like barrier structure in the channel of the nuclear pores, seen in uninfected cells, was missing in the infected cells, giving the impression of fully open pores. Transient expression of poliovirus 2A protease also resulted in relocation of the nuclear proteins. Lysates from poliovirus-infected or 2A-expressing cells induced efflux of 3×EGFP-NLS from the nuclei of permeabilized uninfected cells. This activity was inhibited by the elastase inhibitors elastatinal and
N
-(methoxysuccinyl)-
l
-alanyl-
l
-alanyl-
l
-prolyl-
l
-valine chloromethylketone (drugs known also to be inhibitors of poliovirus protease 2A), a caspase inhibitor zVAD(OMe), fmk, and some other protease inhibitors. These data suggest that 2A elicited nuclear efflux, possibly in cooperation with a zVAD(OMe).fmk-sensitive protease. However, poliovirus infection facilitated nuclear protein efflux also in cells deficient in caspase-3 and caspase-9, suggesting that the efflux may occur without the involvement of these enzymes. The biological relevance of nucleocytoplasmic traffic alterations in infected cells is discussed.
During Lytic Infection Herpes Simplex Virus Type 1 Is Associated with Histones Bearing Modifications That Correlate with Active Transcription
Authors: Kent J. R. , Zeng P.-Y. , Atanasiu D. , Gardner J. , Fraser N. W. , Berger S. L.
Published: August 2004

Herpes simplex virus type 1 (HSV-1) is a large (150-kb) double-stranded DNA virus that forms latent infections in neuronal cells of the human peripheral nervous system. Previous work determined that the HSV-1 genome is found in an ordered nucleosomal structure during latent infection. However, during lytic infection, it was unclear whether viral DNA was in a chromatin state. We examined HSV-1 during lytic infection using micrococcal nuclease digestion and chromatin immunoprecipitation. The HSV-1 genome is at least partially nucleosomal, although apparently not in a regular repeating structure. Analysis of histones associated with HSV-1, within both the promoter and the transcribed regions, revealed covalent amino tail modifications similar to those associated with active host mammalian genes. Certain of the modifications were detected in the temporal order expected of the immediate-early, early, and late gene classes. These data suggest that productive infection may be accompanied by acquisition of a permissive chromatin state.
A Naturally Occurring Variation in the Proline-Rich Region Does Not Attenuate Human Immunodeficiency Virus Type 1 Nef Function
Authors: Rücker Elke , Münch Jan , Wildum Steffen , Brenner Matthias , Eisemann Jutta , Margolis Leonid , Kirchhoff Frank
Published: August 2004

We analyzed human immunodeficiency virus type 1 (HIV-1) Nef variants to further evaluate the functional relevance of the R71T substitution previously proposed to attenuate viral replication (Fackler et al., Curr. Biol. 11:1294-1299, 2001). Our results demonstrate that this variation in the proline-rich region does not significantly affect the functional activity of Nef or HIV-1 infectivity or replication.
Newly Synthesized Hepatitis C Virus Replicon RNA Is Protected from Nuclease Activity by a Protease-Sensitive Factor(s)
Authors: Yang Guang , Pevear Daniel C. , Collett Marc S. , Chunduru Srinivas , Young Dorothy C. , Benetatos Christopher , Jordan Robert
Published: August 2004

Biochemical characterization of hepatitis C virus (HCV) replication using purified, membrane-associated replication complexes is hampered by the presence of endogenous nuclease activity that copurifies with the replication complex. In this study, pulse-chase analyses were used to demonstrate that newly synthesized replicon RNA was protected from nuclease activity by a factor(s) that was sensitive to 0.5% NP-40 or protease treatment. Nuclease susceptibility was not related to disruption of lipid membranes, since NP-40 did not significantly affect the buoyant density of HCV replication complexes or protease susceptibility of HCV NS3 and NS5A proteins. These results suggest that a protease-sensitive factor(s) protects newly synthesized RNA from nuclease degradation.
Detection of JC Virus-Specific Cytotoxic T Lymphocytes in Healthy Individuals
Authors: Du Pasquier R. A. , Schmitz J. E. , Jean-Jacques J. , Zheng Y. , Gordon J. , Khalili K. , Letvin N. L. , Koralnik I. J.
Published: August 2004

The polyomavirus JC (JCV) infects 85% of healthy individuals, and its reactivation in a limited number of immunosuppressed people causes progressive multifocal leukoencephalopathy (PML), a severe demyelinating disease of the central nervous system. We hypothesized that JCV-specific cytotoxic T lymphocytes (CTLs) might control JCV replication in healthy individuals, blocking the evolution of PML. Using51Cr release and tetramer staining assays, we show that 8 of 11 HLA-A*0201+healthy subjects (73%) harbor detectable JCV-specific CD8+CTLs that recognize one or two epitopes of JCV VP1 protein, the HLA-A*0201-restricted VP1p36and VP1p100epitopes. We determined that the frequency of JCV VP1 epitope-specific CTLs varied from less than 1/100,000 to 1/2,494 peripheral blood mononuclear cells. More individuals had JCV VP1-specific than cytomegalovirus-specific CTLs (8 of 11 subjects [73%] versus 2 of 10 subjects [20%], respectively). These results show that a CD8+-T-cell response against JCV is commonly found in immunocompetent people and suggest that these cells might protect against the development of PML.
Effect of the Addition of Oligosaccharides on the Biological Activities and Antigenicity of Influenza A/H3N2 Virus Hemagglutinin
Authors: Abe Yasuhiro , Takashita Emi , Sugawara Kanetsu , Matsuzaki Yoko , Muraki Yasushi , Hongo Seiji
Published: August 2004

Influenza A/H3N2 viruses have developed an increased number of glycosylation sites on the globular head of the hemagglutinin (HA) protein since their appearance in 1968. Here, the effect of addition of oligosaccharide chains to the HA of A/H3N2 viruses on its biological activities was investigated. We constructed seven mutant HAs of A/Aichi/2/68 virus with one to six glycosylation sites on the globular head, as found in natural isolates, by site-directed mutagenesis and analyzed their intracellular transport, receptor binding, and cell fusion activities. The glycosylation sites of mutant HAs correspond to representative A/H3N2 isolates (A/Victoria/3/75, A/Memphis/6/86, or A/Sydney/5/97). The results showed that all the mutant HAs were transported to the cell surface as efficiently as wild-type HA. Although mutant HAs containing three to six glycosylation sites decreased receptor binding activity, their cell fusion activity was not affected. The reactivity of mutant HAs having four to six glycosylation sites with human sera collected in 1976 was much lower than that of wild-type HA. Thus, the addition of new oligosaccharides to the globular head of the HA of A/H3N2 viruses may have provided the virus with an ability to evade antibody pressures by changing antigenicity without an unacceptable defect in biological activity.
Autographa californica
Multiple Nucleopolyhedrovirus
exon0
(
orf141
), Which Encodes a RING Finger Protein, Is Required for Efficient Production of Budded Virus
Authors: Dai Xiaojiang , Stewart Taryn M. , Pathakamuri Joseph A. , Li Qianjun , Theilmann David A.
Published: August 2004

exon0
(
orf141
) of
Autographa californica
multiple nucleopolyhedrovirus (Ac
M
NPV) is a highly conserved baculovirus gene that codes for a predicted 261-amino-acid protein. Located in the C-terminal region of EXON0 are a predicted leucine-rich coiled-coil domain and a RING finger motif. The 5′ 114 nucleotides of
exon0
form part of
ie0
, which is a spliced gene expressed at very early times postinfection, but transcriptional analysis revealed that
exon0
is transcribed as a late gene. To determine the role of
exon0
in the baculovirus life cycle, we used Ac
M
NPV bacmids and generated
exon0
knockout viruses (Ac-exon0-KO) by recombination in
Escherichia coli
. Ac-exon0-KO progressed through the very late phases in Sf9 cells, as evidenced by the development of occlusion bodies in the nuclei of the transfected or infected cells. However, production of budded virus (BV) in Ac-exon0-KO-infected cells was reduced at least 3 orders of magnitude in comparison to that in wild-type virus infection. Microscopy revealed that Ac-exon0-KO was restricted primarily to the cells initially infected, exhibiting a single-cell infection phenotype. Slot blot assays and Western blot analysis indicated that
exon0
deletion did not affect the onset or levels of viral DNA replication or the expression of IE1, IE0, and GP64 prior to BV release. These results demonstrate that
exon0
is required for efficient production of BV in the Ac
M
NPV life cycle but does not affect late occlusion-derived virus.
Mechanism of CD150 (SLAM) Down Regulation from the Host Cell Surface by Measles Virus Hemagglutinin Protein
Authors: Welstead G. Grant , Hsu Eric C. , Iorio Caterina , Bolotin Shelly , Richardson Christopher D.
Published: August 2004

Measles virus has been reported to enter host cells via either of two cellular receptors, CD46 and CD150 (SLAM). CD46 is found on most cells of higher primates, while SLAM is expressed on activated B, T, and dendritic cells and is an important regulatory molecule of the immune system. Previous reports have shown that measles virus can down regulate expression of its two cellular receptors on the host cell surface during infection. In this study, the process of down regulation of SLAM by measles virus was investigated. We demonstrated that expression of the hemagglutinin (H) protein of measles virus was sufficient for down regulation. Our studies provided evidence that interactions between H and SLAM in the endoplasmic reticulum (ER) can promote the down regulation of SLAM but not CD46. In addition, we demonstrated that interactions between H and SLAM at the host cell surface can also contribute to SLAM down regulation. These results indicate that two mechanisms involving either intracellular interactions between H and SLAM in the ER or receptor-mediated binding to H at the surfaces of host cells can lead to the down regulation of SLAM during measles virus infection.
The Conserved Carboxy Terminus of the Capsid Domain of Human Immunodeficiency Virus Type 1 Gag Protein Is Important for Virion Assembly and Release
Authors: Melamed Daniel , Mark-Danieli Michal , Kenan-Eichler Michal , Kraus Osnat , Castiel Asher , Laham Nihay , Pupko Tal , Glaser Fabian , Ben-Tal Nir , Bacharach Eran
Published: August 2004

The retroviral Gag precursor plays an important role in the assembly of virion particles. The capsid (CA) protein of the Gag molecule makes a major contribution to this process. In the crystal structure of the free CA protein of the human immunodeficiency virus type 1 (HIV-1), 11 residues of the C terminus were found to be unstructured, and to date no information exists on the structure of these residues in the context of the Gag precursor molecule. We performed phylogenetic analysis and demonstrated a high degree of conservation of these 11 amino acids. Deletion of this cluster or introduction of various point mutations into these residues resulted in significant impairment of particle infectivity. In this cluster, two putative structural regions were identified, residues that form a hinge region (353-VGGP-356) and those that contribute to an α-helix (357-GHKARVL-363). Overall, mutations in these regions resulted in inhibition of virion production, but mutations in the hinge region demonstrated the most significant reduction. Although all the Gag mutants appeared to have normal Gag-Gag and Gag-RNA interactions, the hinge mutants were characterized by abnormal formation of cytoplasmic Gag complexes. Gag proteins with mutations in the hinge region demonstrated normal membrane association but aberrant rod-like membrane structures. More detailed analysis of these structures in one of the mutants demonstrated abnormal trapped Gag assemblies. These data suggest that the conserved CA C terminus is important for HIV-1 virion assembly and release and define a putative target for drug design geared to inhibit the HIV-1 assembly process.
VP16-Dependent Association of Chromatin-Modifying Coactivators and Underrepresentation of Histones at Immediate-Early Gene Promoters during Herpes Simplex Virus Infection
Authors: Herrera Francisco J. , Triezenberg Steven J.
Published: August 2004

During infection by herpes simplex virus type 1 (HSV-1), the virion protein VP16 activates the transcription of viral immediate-early (IE) genes. Genetic and biochemical assays have shown that the potent transcriptional activation domain of VP16 can associate with general transcription factors and with chromatin-modifying coactivator proteins of several types. The latter interactions are particularly intriguing because previous reports indicate that HSV-1 DNA does not become nucleosomal during lytic infection. In the present work, chemical cross-linking and immunoprecipitation assays were used to probe the presence of activators, general transcription factors, and chromatin-modifying coactivators at IE gene promoters during infection of HeLa cells by wild-type HSV-1 and by RP5, a viral strain lacking the VP16 transcriptional activation domain. The presence of VP16 and Oct-1 at IE promoters did not depend on the activation domain. In contrast, association of RNA polymerase II, TATA-binding protein, histone acetyltransferases (p300 and CBP), and ATP-dependent remodeling proteins (BRG1 and hBRM) with IE gene promoters was observed in wild-type infections but was absent or reduced in cells infected by RP5. In contrast to the previous evidence for nonnucleosomal HSV-1 DNA, histone H3 was found associated with viral DNA at early times of infection. Interestingly, histone H3 was underrepresented on IE promoters in a manner dependent on the VP16 activation domain. Thus, the VP16 activation domain is responsible for recruiting general transcription factors and coactivators to IE promoters and also for dramatically reducing the association of histones with those promoters.
Heat Shock Protein 70 Protects Cells from Cell Cycle Arrest and Apoptosis Induced by Human Immunodeficiency Virus Type 1 Viral Protein R
Authors: Iordanskiy Sergey , Zhao Yuqi , Dubrovsky Larisa , Iordanskaya Tatiana , Chen Mongzhong , Liang Dong , Bukrinsky Michael
Published: August 2004

Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) is an accessory protein that plays an important role in viral pathogenesis. This pathogenic activity of Vpr is related in part to its capacity to induce cell cycle G2arrest and apoptosis of target T cells. A screening for multicopy suppressors of these Vpr activities in fission yeast identified heat shock protein 70 (Hsp70) as a suppressor of Vpr-induced cell cycle arrest. Hsp70 is a member of a family of molecular chaperones involved in innate immunity and protection from environmental stress. In this report, we demonstrate that HIV-1 infection induces Hsp70 in target cells. Overexpression of Hsp70 reduced the Vpr-dependent G2arrest and apoptosis and also reduced replication of the Vpr-positive, but not Vpr-deficient, HIV-1. Suppression of Hsp70 expression by RNA interference (RNAi) resulted in increased apoptosis of cells infected with a Vpr-positive, but not Vpr-defective, HIV-1. Replication of the Vpr-positive HIV-1 was also increased when Hsp70 expression was diminished. Vpr and Hsp70 coimmunoprecipitated from HIV-infected cells. Together, these results identify Hsp70 as a novel anti-HIV innate immunity factor that targets HIV-1 Vpr.
Regulation of Hepatitis C Virus Replication by Interferon Regulatory Factor 1
Authors: Kanazawa Nobuhiko , Kurosaki Masayuki , Sakamoto Naoya , Enomoto Nobuyuki , Itsui Yasuhiro , Yamashiro Tsuyoshi , Tanabe Yoko , Maekawa Shinya , Nakagawa Mina , Chen Cheng-Hsin , Kakinuma Sei , Oshima Shigeru , Nakamura Tetsuya , Kato Takanobu , Wakita Takaji , Watanabe Mamoru
Published: August 2004

Cellular antiviral responses are mediated partly by the expression of interferon-stimulated genes, triggered by viral genomes, their transcripts and replicative intermediates. Persistent replication of a hepatitis C virus (HCV) replicon suggests that the replicon does not elicit cellular innate antiviral responses. In the present study, we investigated regulatory factors of the interferon-mediated antiviral system in cells expressing an HCV replicon. Luciferase reporter assays revealed that the baseline activity of the interferon-stimulated response element (ISRE) was significantly lower in cells harboring the replicon than in naive cells. Among the proteins involved in the IFN/Jak/STAT pathway and in ISRE activity, the expression level of interferon regulatory factor 1 (IRF-1) was found to be significantly lower in cells harboring the replicon. Transfection of an IRF-1 expression construct into cells harboring the replicon caused an increase of ISRE activity, accompanied by suppression of expression of the HCV replicon. Moreover, in cured Huh7 cells from which the HCV replicon had been eliminated, the expression levels of IRF-1 and ISRE activity also were suppressed, demonstrating that the decrease of IRF-1 is attributable, not to active suppression by the viral proteins, but to adaptation of cells that enables replication of the HCV subgenome. The high permissiveness of the cured cells for the replicon was abolished by transgenic supplementation of IRF-1 expression. Taken together, IRF-1 is one of the key host factors that regulate intracellular HCV replication through modulation of interferon-stimulated-gene-mediated antiviral responses.
Inhibition of Cyclooxygenase Activity Reduces Rotavirus Infection at a Postbinding Step
Authors: Rossen John W. A. , Bouma Janneke , Raatgeep Rolien H. C. , Büller Hans A. , Einerhand Alexandra W. C.
Published: August 2004

Elevated levels of prostaglandins (PGs), products of cyclooxygenases (COXs), are found in the plasma and stool of rotavirus-infected children. We sought to determine the role of COXs, PGs, and the signal transduction pathways involved in rotavirus infection to elucidate possible new targets for antiviral therapy. Human intestinal Caco-2 cells were infected with human rotavirus Wa or simian rotavirus SA-11. COX-2 mRNA expression and secreted PGE2levels were determined at different time points postinfection, and the effect of COX inhibitors on rotavirus infection was studied by an immunofluorescence assay (IFA). To reveal the signal transduction pathways involved, the effect of MEK, protein kinase A (PKA), p38 mitogen-activated protein kinase (MAPK), and NF-κB inhibitors on rotavirus infection was analyzed. In infected Caco-2 cells, increased COX-2 mRNA expression and secreted PGE2levels were detected. Indomethacin (inhibiting both COX-1 and COX-2) and specific COX-1 and COX-2 inhibitors reduced rotavirus infection by 85 and 50%, respectively, as measured by an IFA. Indomethacin reduced virus infection at a postbinding step early in the infection cycle, inhibiting virus protein synthesis. Indomethacin did not seem to affect viral RNA synthesis. Inhibitors of MEK, PKA, p38 MAPK, and NF-κB decreased rotavirus infection by at least 40%. PGE2counteracted the effect of the COX and PKA inhibitors but not of the MEK, p38 MAPK, and NF-κB inhibitors. Conclusively, COXs and PGE2are important mediators of rotavirus infection at a postbinding step. The ERK1/2 pathway mediated by PKA is involved in COX induction by rotavirus infection. MAPK and NF-κB pathways are involved in rotavirus infection but in a PGE2-independent manner. This report offers new perspectives in the search for therapeutic agents in treatment of severe rotavirus-mediated diarrhea in children.
Integral Membrane Protein P16 of Bacteriophage PRD1 Stabilizes the Adsorption Vertex Structure
Authors: Jaatinen Silja T. , Viitanen Salla J. , Bamford Dennis H. , Bamford Jaana K. H.
Published: August 2004

The icosahedral membrane-containing double-stranded DNA bacteriophage PRD1 has a labile receptor binding spike complex at the vertices. This complex, which is analogous to that of adenovirus, is formed of the penton protein P31, the spike protein P5, and the receptor binding protein P2. Upon infection, the internal phage membrane transforms into a tubular structure that protrudes through a vertex and penetrates the cell envelope for DNA injection. We describe here a new class of PRD1 mutants lacking virion-associated integral membrane protein P16. P16 links the spike complex to the viral membrane and is necessary for spike stability. We also show that the unique vertex used for DNA packaging is intact in the P16-deficient particle, indicating that the 11 adsorption vertices and the 1 portal vertex are functionally and structurally distinct.
NSs Protein of Rift Valley Fever Virus Blocks Interferon Production by Inhibiting Host Gene Transcription
Authors: Billecocq Agnès , Spiegel Martin , Vialat Pierre , Kohl Alain , Weber Friedemann , Bouloy Michèle , Haller Otto
Published: August 2004

Rift Valley fever virus (RVFV) is an important cause of epizootics and epidemics in Africa and a potential agent of bioterrorism. A better understanding of the factors that govern RVFV virulence and pathogenicity is required, given the urgent need for antiviral therapies and safe vaccines. We have previously shown that RVFV strains with mutations in the NSs gene are excellent inducers of α/β interferon (IFN-α/β) and are highly attenuated in mice. Here, we demonstrate that NSs is sufficient to block IFN-β gene expression at the transcriptional level. In cells transiently expressing NSs, IFN-β transcripts were not inducible by viral infection or by transfection of poly(I:C). NSs with anti-IFN activity accumulated in the nucleus. In contrast, mutant forms of NSs that had lost their IFN-inhibiting activity remained in the cytoplasm, indicating that nuclear localization plays a role. IFN synthesis is regulated by specific transcription factors, including interferon regulatory factor (IRF-3), NF-κB, and AP-1. In the presence of NSs, IRF-3 was still activated and moved to the nucleus. Likewise, NF-κB and AP-1 were activated normally, as shown in electrophoretic mobility shift assays. Moreover, NSs was found to inhibit transcriptional activity of a constitutive promoter, in agreement with recent findings showing that NSs targets the basal cellular transcription factor TFIIH. The present results suggest that NSs, unlike other viral IFN antagonists, does not inhibit IFN-specific transcription factors but blocks IFN gene expression at a subsequent step.
Tagging Retrovirus Vectors with a Metal Binding Peptide and One-Step Purification by Immobilized Metal Affinity Chromatography
Authors: Ye Kaiming , Jin Sha , Ataai Mohammad M. , Schultz Jerome S. , Ibeh Jeanette
Published: August 2004

Retroviral vectors produced from packaging cells are invariably contaminated by protein, nucleic acid, and other substances introduced in the manufacturing process. Elimination of these contaminants from retroviral vector preparations is helpful to reduce unwanted side effects, and purified vector preparations are desirable to improve reproducibility of therapeutic effect. Here we report a novel approach to engineer a metal binding peptide (MBP)-tagged murine leukemia virus (MuLV), allowing for one-step purification of retroviral vectors by immobilized metal affinity chromatography (IMAC). We inserted a His6peptide into an ecotropic envelope protein (Env) by replacing part of its hypervariable region sequence with a sequence encoding the His6peptide. Display of the His6tag on the surface of Env endowed the vectors with a high affinity for immobilized metal ions, such as nickel. We demonstrated that the His6-tagged MuLV could be produced to high titers and could be highly purified by one-step IMAC. The protein and DNA contaminants in the purified vector supernatants were below 7 μg/ml and 25 pg/ml, respectively, indicating a 1,229-fold reduction in protein contaminant level and a 6,800-fold reduction in DNA contaminant level. About 56% of the viral vectors were recovered in the IMAC purification. The purified vectors retained their functionality and infectivity. These results establish that an MBP can be functionally displayed on the surface of ecotropic retroviruses without interfering with their integrity, and MBP-tagged retroviral vectors can be highly purified by one-step IMAC.