bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual Mechanisms of Pestiviral Superinfection Exclusion at Entry and RNA Replication
Authors: Lee Young-Min , Tscherne Donna M. , Yun Sang-Im , Frolov Ilya , Rice Charles M.
Published: February 2005

For many viruses, primary infection has been shown to prevent superinfection by a homologous second virus. In this study, we investigated superinfection exclusion of bovine viral diarrhea virus (BVDV), a positive-sense RNA pestivirus. Cells acutely infected with BVDV were protected from superinfection by homologous BVDV but not with heterologous vesicular stomatitis virus. Superinfection exclusion was established within 30 to 60 min but was lost upon passaging of persistently infected cells. Superinfecting BVDV failed to deliver a translatable genome into acutely infected cells, indicating a block in viral entry. Deletion of structural protein E2 from primary infecting BVDV abolished this exclusion. Bypassing the entry block by RNA transfection revealed a second block at the level of replication but not translation. This exclusion did not require structural protein expression and was inversely correlated with the level of primary BVDV RNA replication. These findings suggest dual mechanisms of pestivirus superinfection exclusion, one at the level of viral entry that requires viral glycoprotein E2 and a second at the level of viral RNA replication.
E1A-CR3 Interaction-Dependent and -Independent Functions of mSur2 in Viral Replication of Early Region 1A Mutants of Mouse Adenovirus Type 1
Authors: Fang Lei , Spindler Katherine R.
Published: February 2005

mSur2, a subunit of the Mediator complex, is required for efficient mouse adenovirus type 1 (MAV-1) replication (L. Fang, J. L. Stevens, A. J. Berk, and K. R. Spindler, J. Virol.78:12888-12900, 2004). We examined the contributions of early-region 1A (E1A) to mSur2 function in MAV-1 replication with E1A mutant viruses. At a multiplicity of infection (MOI) of 1, viruses containing CR3 replicated better in Sur2+/+mouse embryonic fibroblasts (MEFs) than in Sur2−/−MEFs. In contrast, viruses lacking CR3 replicated no better in Sur2+/+than in Sur2−/−MEFs. This result supports the hypothesis that the E1A CR3-mSur2 interaction is important for MAV-1 replication. However, at an MOI of 0.05, viruses lacking CR3 showed replication defects in Sur2−/−MEFs compared to Sur2+/+MEFs, suggesting an E1A CR3 interaction-independent function of mSur2 in MAV-1 replication in cell culture. Paradoxically, CR1Δ, CR2Δ, and CR3Δ mutant viruses replicated slightly more efficiently than wild-type (wt) MAV-1 and E1A null mutant viruses in Sur2−/−MEFs at an MOI of 0.05. Coinfection of Sur2−/−MEFs with wt MAV-1 and CR1Δ, CR2Δ, or CR3Δ mutant viruses rescued the defects of wt MAV-1 replication. This result suggests that an inhibiting effect on wt E1A protein expression and/or E1A function might account for the severe viral replication defect of MAV-1 in Sur2−/−MEFs at an MOI of 0.05. Moreover, titrations of virus yields from infected brains of inbred strains of mice showed that E1A null and CR3Δ mutant viruses had a significant defect in virus replication compared to wt MAV-1. This result supports the hypothesis that the MAV-1 E1A-mSur2 interaction is important in MAV-1 replication in mice.
Recombinant Herpes Simplex Virus Type 1 (HSV-1) Codelivering Interleukin-12p35 as a Molecular Adjuvant Enhances the Protective Immune Response against Ocular HSV-1 Challenge
Authors: Osorio Yanira , Ghiasi Homayon
Published: February 2005

An important aspect of ocular herpes simplex virus type 1 (HSV-1) vaccine development is identification of an appropriate adjuvant capable of significantly reducing both virus replication in the eye and explant reactivation in trigeminal ganglia. We showed recently that a recombinant HSV-1 vaccine expressing interleukin-4 (IL-4) is more efficacious against ocular HSV-1 challenge than recombinant viruses expressing IL-2 or gamma interferon (IFN-γ) (Y. Osorio and H. Ghiasi, J. Virol. 77:5774-5783, 2003). We have now constructed and compared recombinant HSV-1 viruses expressing IL-12p35 or IL-12p40 molecule with IL-4-expressing HSV-1 recombinant virus. BALB/c mice were immunized intraperitoneally with IL-12p35-, IL-12p40-, IL-12p35+IL-12p40-, or IL-4-expressing recombinant HSV-1 viruses. Controls included mice immunized with parental virus and mice immunized with the avirulent strain KOS. The efficacy of each vaccine in protecting against ocular challenge with HSV-1 was assessed in terms of survival, eye disease, virus replication in the eye, and explant reactivation. Neutralizing antibody titers, T-cell responses, and expression of 32 cytokines and chemokines were also evaluated. Mice immunized with recombinant HSV-1 expressing IL-12p35 exhibited the lowest virus replication in the eye, the most rapid virus clearance, and the lowest level of explant reactivation. The higher efficacy against ocular virus replication and explant reactivation correlated with higher neutralizing antibody titers, cytotoxic-T-lymphocyte activities, and IFN-γ expression in recombinant HSV-1 expressing IL-12p35 compared to other vaccines. Mice immunized with both IL-12p35 and IL-12p40 had lower neutralizing antibody responses than mice immunized with IL-12p35 alone. Our results confirm that recombinant virus vaccines expressing cytokine genes can enhance the overall protection against infection, with the IL-12p35 vaccine being the most efficacious of those tested. Collectively, the results support the potential use of IL-12p35 as a vaccine adjuvant, without the toxicity-associated concerns of IL-12.
Modulation of the CD8+-T-Cell Response by CD4+CD25+Regulatory T Cells in Patients with Hepatitis B Virus Infection
Authors: Franzese Ornella , Kennedy Patrick T. F. , Gehring Adam J. , Gotto Jim , Williams Roger , Maini Mala K. , Bertoletti Antonio
Published: February 2005

CD4+CD25+regulatory T cells have been shown to maintain peripheral tolerance against self and foreign antigens. In this study we analyzed the effect of circulating CD4+CD25+T cells on CD8+-T-cell responses of patients with chronic and resolved hepatitis B virus (HBV) infection. We demonstrated that circulating CD4+CD25+T cells modulate the function and expansion of HBV-specific CD8+cells ex vivo in all patients, regardless of whether they have chronic or resolved HBV infection. The possible role of CD4+CD25+T cells in the pathogenesis of chronic HBV infection is not supported by these data. However, these results might have implications for optimizing future immunotherapeutic approaches to HBV treatment.
Altered Function in CD8+T Cells following Paramyxovirus Infection of the Respiratory Tract
Authors: Gray Peter M. , Arimilli Subhashini , Palmer Ellen M. , Parks Griffith D. , Alexander-Miller Martha A.
Published: February 2005

For many respiratory pathogens, CD8+T cells have been shown to play a critical role in clearance. However, there are still many unanswered questions with regard to the factors that promote the most efficacious immune response and the potential for immunoregulation of effector cells at the local site of infection. We have used infection of the respiratory tract with the model paramyxovirus simian virus 5 (SV5) to study CD8+T-cell responses in the lung. For the present study, we report that over time a population of nonresponsive, virus-specific CD8+T cells emerged in the lung, culminating in a lack of function in ∼85% of cells specific for the immunodominant epitope from the viral matrix (M) protein by day 40 postinfection. Concurrent with the induction of nonresponsiveness, virus-specific cells that retained function at later times postinfection exhibited an increased requirement for CD8 engagement. This change was coupled with a nearly complete loss of functional phosphoprotein-specific cells, a response previously shown to be almost exclusively CD8 independent. These studies add to the growing evidence for immune dysregulation following viral infection of the respiratory tract.
Differential Role for TLR3 in Respiratory Syncytial Virus-Induced Chemokine Expression
Authors: Rudd Brian D. , Burstein Ezra , Duckett Colin S. , Li Xiaoxia , Lukacs Nicholas W.
Published: February 2005

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in young infants worldwide. Previous studies have reported that the induction of interleukin-8/CXCL8 and RANTES/CCL5 correlates with disease severity in humans. The production of these chemokines is elicited by viral replication and is NF-κB dependent. RSV, a negative-sense single-stranded RNA virus, requires full-length positive-sense RNA for synthesis of new viral RNA. The aim of our studies was to investigate whether active viral replication by RSV could evoke chemokine production through TLR3-mediated signaling pathways. In TLR3-transfected HEK 293 cells, live RSV preferentially activated chemokines in both a time- and dose-dependent manner compared to vector controls. RSV was also shown to upregulate TLR3 in human lung fibroblasts and epithelial cells (MRC-5 and A549). Targeting the expression of TLR3 with small interfering RNA decreased synthesis of IP-10/CXCL10 and CCL5 but did not significantly reduce levels of CXCL8. Blocking the expression of the adapter protein MyD88 established a role for MyD88 in CXCL8 production, whereas CCL5 synthesis was found to be MyD88 independent. Production of CCL5 by RSV was induced directly through TLR3 signaling pathways and did not require interferon (IFN) signaling through the IFN-α/β receptor. TLR3 did not affect viral replication, since equivalent viral loads were recovered from RSV-infected cells despite altered TLR3 expression. Taken together, our studies indicate that TLR3 mediates inflammatory cytokine and chemokine production in RSV-infected epithelial cells.
B-Cell Responses in Patients Who Have Recovered from Severe Acute Respiratory Syndrome Target a Dominant Site in the S2 Domain of the Surface Spike Glycoprotein
Authors: Zhong Xiaofen , Yang Huanghao , Guo Zu-Feng , Sin Wan-Yee Fion , Chen Wei , Xu Junjie , Fu Ling , Wu Jie , Mak Chun-Kit Gannon , Cheng Chak-Sum Samuel , Yang Yanzhen , Cao Shuyong , Wong Tin-Yau , Lai Sik-To , Xie Yong , Guo Zhihong
Published: February 2005

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel strain of coronavirus. Examination of the immune responses of patients who have recovered from SARS should provide important information for design of a safe and effective vaccine. We determined the continuous viral epitopes targeted by antibodies in plasma samples from convalescent SARS patients through biopanning with a vast M13 phage display dodecapeptide library. These epitopes converged to very short peptide fragments, one on each of the structural proteins spike and nucleocapsid and the nonstructural proteins 3a, 9b, and nsp 3. Immunoassays found that most of the patients who had recovered from SARS developed complementary antibodies to the epitope-rich region on the spike S2 protein, indicating that this is an immunodominant site on the viral envelope comprising the spike, matrix, and small envelope glycoproteins. These S2-targeting antibodies were shown to effectively neutralize the coronavirus, indicating that they provided protective immunity to help the patients recover from the viral infection. These results suggest that the SARS coronavirus might have an antigenic profile distinct from those of other human or animal coronaviruses. Due to the tested safety and protective effects of the convalescent-phase serological antibodies, identification of their complementary antigens may enable the design of an epitope-based vaccine to prevent potential antibody-mediated immunuopathology.
Characterization of an In Vitro Model of Alphavirus Infection of Immature and Mature Neurons
Authors: Vernon Patty S. , Griffin Diane E.
Published: February 2005

Terminally differentiated, mature neurons are essential cells that are not easily regenerated. Neurotropic viruses, such as Sindbis virus (SV), cause encephalomyelitis through their ability to replicate in neurons. SV causes the death of immature neurons, while mature neurons can often survive infection. The lack of a reproducible and convenient neuronal cell culture system has hindered a detailed study of the differences in levels of virus replication between immature and mature neurons and the molecular events involved in virus clearance from mature neurons. We have characterized SV replication in immortalized CSM14.1 rat neuronal cells that can be differentiated into neurons. During differentiation, CSM14.1 cells ceased dividing, developed neuronal morphology, and expressed neuron-specific cell markers. SV infection of undifferentiated CSM14.1 cells was efficient and resulted in high levels of virus replication and cell death. SV infection of differentiated CSM14.1 cells was less efficient and resulted in the production of 10- to 100-fold less virus and cell survival. In undifferentiated cells, SV induced a rapid shutdown of cellular protein synthesis and pE2 was efficiently processed to E2 (ratio of E2 to pE2, 2.14). In differentiated cells, the SV-induced shutdown of cellular protein synthesis was transient and pE2 was the primary form of E2 in cells (ratio of E2 to pE2, 0.0426). We conclude that age-dependent restriction of virus replication is an intrinsic property of maturing neurons and that the CSM14.1 cell line is a convenient model system for investigating the interactions of alphaviruses with neurons at various stages of differentiation.
Kaposi's Sarcoma-Associated Herpesvirus Reactivation Is Regulated by Interaction of Latency-Associated Nuclear Antigen with Recombination Signal Sequence-Binding Protein Jκ, the Major Downstream Effector of the Notch Signaling Pathway
Authors: Lan Ke , Kuppers Daniel A. , Robertson Erle S.
Published: February 2005

Kaposi's sarcoma-associated herpesvirus (KSHV) is the major biological cofactor contributing to development of Kaposi's sarcoma. KSHV establishes a latent infection in human B cells expressing the latency-associated nuclear antigen (LANA), a critical factor in the regulation of viral latency. LANA controls KSHV latent infection through repression of RTA, an activator of many lytic promoters. RTA activates the expression of several lytic viral genes by interacting with recombination signal sequence-binding protein Jκ (RBP-Jκ), a transcriptional repressor and the target of the Notch signaling pathway. The recognition that a number of KSHV lytic gene promoters, including RTA, contain RBP-Jκ binding sites raised the possibility that RBP-Jκ-mediated repression may be central to the establishment of latency. Here, we tested this hypothesis by examining the regulation of RTA by LANA through binding to RBP-Jκ. This study demonstrates that LANA physically associates with RBP-Jκ in vitro and in KSHV-infected cells, with the complex formed capable of binding to RBP-Jκ cognate sequences. RBP-Jκ binding sites within the RTA promoter have been found to be critical for LANA-mediated repression. Our study describes a novel mechanism through which LANA maintains KSHV latency by targeting a major downstream effector of the Notch signaling pathway.
Receptor-Induced Conformational Changes in the SU Subunit of the Avian Sarcoma/Leukosis Virus A Envelope Protein: Implications for Fusion Activation
Authors: Delos Sue E. , Godby Jesse A. , White Judith M.
Published: February 2005

The avian sarcoma/leukosis virus (ASLV) is activated for fusion by a two-step mechanism. For ASLV subgroup A (ASLV-A), association with its receptor (Tva) at neutral pH converts virions to a form that can bind target membranes and, in some assays, induce the lipid-mixing stage of fusion. Low pH is necessary to complete the fusion reaction. ASLV-A env (EnvA) exists on the viral surface as a trimer of heterodimers consisting of receptor binding (SU-A) and fusion-mediating (TM-A) subunits. As the receptor binding and fusion-mediating functions reside in separate subunits, we hypothesize that SU-A and TM-A are conformationally coupled. To begin to understand the effect of the binding of a soluble 47-residue domain of the receptor (sTva) on this coupling and the subsequent function of low pH, we prepared recombinant proteins representing full-length SU-A and a nested set of deletion mutant proteins. Full-length SU-A binds sTva with high affinity, but even small deletions at either the N or the C terminus severely impair sTva binding. We have purified the full-length SU-A subunit and characterized its interactions with sTva and the subsequent effect of low pH on the complex. sTva binds SU-A with an apparent

K
D

of 3 pM. Complex formation occludes hydrophobic surfaces and tryptophan residues and leads to a partial loss of α-helical structure in SU-A. Low pH does not alter the off rate for the complex, further alter the secondary structure of SU-A, or induce measurable changes in tryptophan environment. The implications of these findings for fusion are discussed.
Stoichiometry of Antibody Neutralization of Human Immunodeficiency Virus Type 1
Authors: Yang Xinzhen , Kurteva Svetla , Lee Sandra , Sodroski Joseph
Published: February 2005

The human immunodeficiency virus envelope glycoproteins function as trimers on the viral surface, where they are targeted by neutralizing antibodies. Different monoclonal antibodies neutralize human immunodeficiency virus type 1 (HIV-1) infectivity by binding to structurally and functionally distinct moieties on the envelope glycoprotein trimer. By measuring antibody neutralization of viruses with mixtures of neutralization-sensitive and neutralization-resistant envelope glycoproteins, we demonstrate that the HIV-1 envelope glycoprotein trimer is inactivated by the binding of a single antibody molecule. Virus neutralization requires essentially all of the functional trimers to be occupied by at least one antibody. This model applies to antibodies differing in neutralizing potency and to virus isolates with various neutralization sensitivities. Understanding these requirements for HIV-1 neutralization by antibodies will assist in establishing goals for an effective AIDS vaccine.
The Cytokine Osteopontin Modulates the Severity of Rotavirus Diarrhea
Authors: Rollo Ellen E. , Hempson Scott J. , Bansal Ajay , Tsao Ernest , Habib Iman , Rittling Susan R. , Denhardt David T. , Mackow Erich R. , Shaw Robert D.
Published: February 2005

Osteopontin (OPN) is a sialated phosphoprotein found in tissues and secreted into body fluids. It is an integrin ligand with pleiotropic functions as an extracellular matrix protein in mineralized tissues and a cytokine that is active in cell signaling (A. B. Tuck, C. Hota, S. M. Wilson, and A. F. Chambers, Oncogene22:1198-1205, 2003). To determine whether OPN may be important in mucosal defense against viral pathogens, we evaluated the OPN response to rotavirus infection and the extent of diarrhea manifested by infectedopnnull mutant (opn−/−) mice. Reverse transcription-PCR, Northern and Western blots, and immunohistochemical studies of the HT-29 intestinal epithelial cell line and murine intestine were used to evaluate OPN mRNA and product. Intestinal closed loops and diarrheal observations determined disease severity and duration. OPN mRNA levels increased after infection of HT-29 cells, peaking in 4 to 6 h. Infected cultures contained 925 μg of OPN/ml, while for controls the levels were below detection (50 μg/ml). Infection increased OPN mRNA levels in intestinal tissue between 2 and 24 h postinoculation and increased OPN protein in intestinal fluid. The cellular localization of OPN was supranuclear and apical, and responding cells were diffusely distributed on the villus surface. Three days after infection, closed intestinal loops fromopn−/−mice contained more fluid than loops from controls, although secretion levels at the onset of illness were similar. Null mutant mice experienced more intense and prolonged diarrhea than controls. Rotavirus infection of intestinal epithelial cells and murine intestine caused marked increases in OPN mRNA levels and secreted OPN protein. OPN-deficient mice suffered prolonged disease.
Increased Multinucleoside Drug Resistance and Decreased Replicative Capacity of a Human Immunodeficiency Virus Type 1 Variant with an 8-Amino-Acid Insert in the Reverse Transcriptase
Authors: van der Hoek Lia , Back Nicole , Jebbink Maarten F. , de Ronde Anthony , Bakker Margreet , Jurriaans Suzanne , Reiss Peter , Parkin Neil , Berkhout Ben
Published: February 2005

Resistance to antiretroviral drugs is generally conferred by specific amino acid substitutions, rather than insertions or deletions, in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The exception to these findings is the amino acid insertions found in the β3-β4 loop of the RT enzyme in response to treatment with nucleoside reverse transcriptase inhibitors. This insert consists most commonly of two amino acids, but we describe in detail the evolution of a variant with an 8-amino-acid (aa) insert in a patient treated with zidovudine (ZDV) and 2′-3′-dideoxycytidine (ddC). The 24-nucleotide insert is a partial duplication of local sequences but also contains a sequence segment of unknown origin. Extensive sequence analysis of longitudinal patient samples indicated that the HIV-1 population prior to the start of therapy contained not the wild-type amino acid 215T in RT but a mixture with 215D and 215C. Treatment with ZDV and subsequent ZDV-ddC combination therapy resulted in the evolution of an HIV-1 variant with a typical ZDV resistance genotype (41L, 44D, 67N, 69D, 210W, 215Y), which was slowly replaced by the insert-containing variant (41L, 44D, insert at position 69, 70R, 210W, 215Y). The latter variant demonstrated increased resistance to a wide range of drugs, indicating that the 8-aa insert augments nucleoside analogue resistance. The gain in drug resistance of the insert variant came at the expense of a reduction in replication capacity when assayed in the absence of drugs. We compared these data with the resistance and replication properties of 133 insert-containing sequences of different individuals present in the ViroLogic database and found that the size and actual sequence of the insert at position 69 influence the level of resistance to nucleoside analogues.
Stable Secondary Structure near the Nicking Site for Adeno-Associated Virus Type 2 Rep Proteins on Human Chromosome 19
Authors: Jang Ming Y. , Yarborough OrLando H. , Conyers Gary B. , McPhie Peter , Owens Roland A.
Published: February 2005

Adeno-associated virus serotype 2 (AAV-2) can preferentially integrate its DNA into a 4-kb region of human chromosome 19, designated
AAVS1
. The nicking activity of AAV-2's Rep68 or Rep78 proteins is essential for preferential integration. These proteins nick at the viral origin of DNA replication and at a similar site within
AAVS1
. The current nicking model suggests that the strand containing the nicking site is separated from its complementary strand prior to nicking. In AAV serotypes 1 through 6, the nicking site is flanked by a sequence that is predicted to form a stem-loop with standard Watson-Crick base pairing. The region flanking the nicking site in
AAVS1
(5′-GGCGGCGGT/TGGGGCTCG-3′ [the slash indicates the nicking site]) lacks extensive potential for Watson-Crick base pairing. We therefore performed an empirical search for a stable secondary structure. By comparing the migration of radiolabeled oligonucleotides containing wild-type or mutated sequences from the
AAVS1
nicking site to appropriate standards, on native and denaturing polyacrylamide gels, we have found evidence that this region forms a stable secondary structure. Further confirmation was provided by circular dichroism analyses. We identified six bases that appear to be important in forming this putative secondary structure. Mutation of five of these bases, within the context of a double-stranded nicking substrate, reduces the ability of the substrate to be nicked by Rep78 in vitro. Four of these five bases are outside the previously recognized GTTGG nicking site motif and include parts of the CTC motif that has been demonstrated to be important for integration targeting.
Analysis of Human Cytomegalovirus
ori
Lyt Sequence Requirements in the Context of the Viral Genome
Authors: Borst Eva-Maria , Messerle Martin
Published: February 2005

During the lytic phase of infection, replication of herpesvirus genomes initiates at the lytic origin of replication,
ori
Lyt. Many herpesviruses harbor more than one lytic origin, but so far, only one
ori
Lyt has been identified for human cytomegalovirus (HCMV). Evidence for the existence of additional lytic origins of HCMV has remained elusive. On the basis of transient replication assays with cloned viral fragments, HCMV
ori
Lyt was described as a core region of 1.5 kbp (minimal
ori
Lyt) flanked by auxiliary sequences required for maximal replication activity (complete
ori
Lyt). It remained unclear whether minimal
ori
Lyt alone can drive the replication of HCMV in the absence of its accessory regions. To investigate the sequence requirements of
ori
Lyt in the context of the viral genome, mutant genomes were constructed lacking either minimal or complete
ori
Lyt. These genomes were not infectious, suggesting that HCMV contains only one lytic origin of replication. Either minimal or complete
ori
Lyt was then ectopically reinserted into the
ori
Lyt-depleted genomes. Only the mutant genomes carrying complete
ori
Lyt led to infectious progeny. Remarkably, inversion of the 1.5-kbp core origin relative to its flanking regions resulted in a replication-defective genome. Mutant genomes carrying minimal
ori
Lyt plus the left flanking region gave rise to minifoci, but genomes harboring minimal
ori
Lyt together with the right flanking region were noninfectious. We conclude that the previously defined minimal lytic origin is not sufficient to drive replication of the HCMV genome. Rather, our results underline the importance of the accessory regions and their correct arrangement for the function of HCMV
ori
Lyt.
Long-Term Cytomegalovirus Infection Leads to Significant Changes in the Composition of the CD8+T-Cell Repertoire, Which May Be the Basis for an Imbalance in the Cytokine Production Profile in Elderly Persons
Authors: Almanzar Giovanni , Schwaiger Susanne , Jenewein Brigitte , Keller Michael , Herndler-Brandstetter Dietmar , Würzner Reinhard , Schönitzer Diether , Grubeck-Loebenstein Beatrix
Published: February 2005

In spite of the present belief that latent cytomegalovirus (CMV) infection drives CD8+T-cell differentiation and induces premature immune senescence, no systematic studies have so far been performed to compare phenotypical and functional changes in the CD8+T-cell repertoire in CMV-infected and noninfected persons of different age groups. In the present study, number, cytokine production, and growth potential of naïve (CD45RA+CD28+), memory (CD45RA−CD28+), and effector (CD45RA+CD28−or CD45RA−CD28−) CD8+T cells were analyzed in young, middle-aged, and elderly clinically healthy persons with a positive or negative CMV antibody serology. Numbers and functional properties of CMVpp65495-503-specific CD8+T cells were also studied. We demonstrate that aging as well as CMV infection lead to a decrease in the size of the naïve CD8+T-cell pool but to an increase in the number of CD8+effector T cells, which produce gamma interferon but lack substantial growth potential. The size of the CD8+memory T-cell population, which grows well and produces interleukin-2 (IL-2) and IL-4, also increases with aging, but this increase is missing in CMV carriers. Life-long latent CMV infection seems thus to diminish the size of the naïve and the early memory T-cell pool and to drive a Th1 polarization within the immune system. This can lead to a reduced diversity of CD8 responses and to chronic inflammatory processes which may be the basis of severe health problems in elderly persons.
Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions
Authors: Fujii Ken , Fujii Yutaka , Noda Takeshi , Muramoto Yukiko , Watanabe Tokiko , Takada Ayato , Goto Hideo , Horimoto Taisuke , Kawaoka Yoshihiro
Published: February 2005

The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3′ coding region are critical for efficient NS vRNA incorporation and that deletion of the 3′ segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3′ NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3′ end) form a structure that is required for efficient influenza A virus vRNA packaging.
Identification of an Essential Domain in the Herpes Simplex Virus 1 U
L
34 Protein That Is Necessary and Sufficient To Interact with U
L
31 Protein
Authors: Liang Li , Baines Joel D.
Published: February 2005

Previous results have indicated that the herpes simplex virus 1 U
L
31 and U
L
34 proteins interact and form a complex at the inner nuclear membranes of infected cells, where both play important roles in the envelopment of nucleocapsids at the inner nuclear membrane. In the work described here, mapping studies using glutathione
S
-transferase pull-down assays indicated that amino acids 137 to 181 of the U
L
34 protein are sufficient to mediate an interaction with the U
L
31 protein. A recombinant virus (v3480) lacking U
L
34 codons 138 to 181 was constructed. Similar to a U
L
34 null virus, v3480 failed to replicate on Vero cells and grew to a limited extent on rabbit skin cells. A U
L
34-expressing cell line restored v3480 growth and plaque formation. Similar to the localization of U
L
31 protein in cells infected with a U
L
34 null virus, the U
L
31 protein was present in the nuclei of Hep2 cells infected with v3480. Hep2 cells infected with v3480 contained the U
L
34 protein in the cytoplasm, the nucleus, and the nuclear membrane, and this was noted to be similar to the appearance of cells infected with a U
L
31 null virus. In transient expression assays, the interaction between U
L
34 amino acids 137 to 181 and the U
L
31 protein was sufficiently robust to target green fluorescent protein and emerin to intranuclear sites that contained the U
L
31 protein. These data indicate that amino acids 137 to 181 of the U
L
34 protein are (i) sufficient to mediate interactions with the U
L
31 protein in vitro and in vivo, (ii) necessary for the colocalization of U
L
31 and U
L
34 in infected cells, and (iii) essential for normal viral replication.
Complementarity in the Supramolecular Design of Arenaviruses and Retroviruses Revealed by Electron Cryomicroscopy and Image Analysis
Authors: Neuman Benjamin W. , Adair Brian D. , Burns John W. , Milligan Ronald A. , Buchmeier Michael J. , Yeager Mark
Published: February 2005

Arenaviruses are rodent-borne agents of diseases, including potentially lethal human hemorrhagic fevers. These enveloped viruses encapsidate a bisegmented ambisense single-stranded RNA genome that can be packaged in variable copy number. Electron cryomicroscopy and image analysis of New World Pichinde and Tacaribe arenaviruses and Old World lymphocytic choriomeningitis virus revealed pleomorphic enveloped particles ranging in diameter from ∼400 to ∼2,000 Å. The surface spikes were spaced ∼100 Å apart and extended ∼90 Å from the maximum phospholipid headgroup density of the outer bilayer leaflet. Distinctive stalk and head regions extended radially ∼30 and ∼60 Å from the outer bilayer leaflet, respectively. Two interior layers of density apposed to the inner leaflet of the viral lipid bilayer were assigned as protein Z and nucleoprotein (NP) molecules on the basis of their appearance, spacing, and projected volume. Analysis of en face views of virions lacking the GP-C spikes showed reflections consistent with paracrystalline packing of the NP molecules in a lattice with edges of ∼57 and ∼74 Å. The structural proteins of retroviruses and arenaviruses assemble with similar radial density distributions, using common cellular components.
Deletion of the Second Immunoglobulin-Like Domain of Nectin-1 Alters Its Intracellular Processing and Localization and Ability To Mediate Entry of Herpes Simplex Virus
Authors: Struyf Frank , Plate Aileen E. , Spear Patricia G.
Published: February 2005

Nectin-1 is an immunoglobulin (Ig)-like entry receptor for herpes simplex virus (HSV). Like other nectins, nectin-1 forms dimers and mediates cell adhesion through interactions with other nectins. We constructed a second-domain deletion mutant of nectin-1 (nectin-1-Δ2) to examine the role of the second Ig-like domain in HSV entry. Nectin-1-Δ2 exhibited a severely reduced ability to mediate HSV entry and accumulated in the endoplasmic reticulum but retained the ability to interact with its HSV ligand, gD. The failure of nectin-1-Δ2 to mediate HSV entry probably resulted from its failure to be transported to a membrane targeted by HSV for viral entry.
Lambda Interferon Inhibits Hepatitis B and C Virus Replication
Authors: Robek Michael D. , Boyd Bryan S. , Chisari Francis V.
Published: February 2005

Lambda interferon (IFN-λ) induces an intracellular IFN-α/β-like antiviral response through a receptor complex distinct from the IFN-α/β receptor. We therefore determined the ability of IFN-λ to inhibit hepatitis B virus (HBV) and hepatitis C virus (HCV) replication. IFN-λ inhibits HBV replication in a differentiated murine hepatocyte cell line with kinetics and efficiency similar to IFN-α/β and does not require the expression of IFN-α/β or IFN-γ. Furthermore, IFN-λ blocked the replication of a subgenomic and a full-length genomic HCV replicon in human hepatocyte Huh7 cells. These results suggest the possibility that IFN-λ may be therapeutically useful in the treatment of chronic HBV or HCV infection.
OPTIMIZING LONG INTRINSIC DISORDER PREDICTORS WITH PROTEIN EVOLUTIONARY INFORMATION
Authors: PENG KANG , VUCETIC SLOBODAN , RADIVOJAC PREDRAG , BROWN CELESTE J. , DUNKER A. KEITH , OBRADOVIC ZORAN
Published: February 2005

Protein existing as an ensemble of structures, called intrinsically disordered, has been shown to be responsible for a wide variety of biological functions and to be common in nature. Here we focus on improving sequence-based predictions of long (>30 amino acid residues) regions lacking specific 3-D structure by means of four new neural-network-based Predictors Of Natural Disordered Regions (PONDRs): VL3, VL3H, VL3P, and VL3E. PONDR VL3 used several features from a previously introduced PONDR VL2, but benefitted from optimized predictor models and a slightly larger (152 vs. 145) set of disordered proteins that were cleaned of mislabeling errors found in the smaller set. PONDR VL3H utilized homologues of the disordered proteins in the training stage, while PONDR VL3P used attributes derived from sequence profiles obtained by PSI-BLAST searches. The measure of accuracy was the average between accuracies on disordered and ordered protein regions. By this measure, the 30-fold cross-validation accuracies of VL3, VL3H, and VL3P were, respectively, 83.6 ± 1.4%, 85.3 ± 1.4%, and 85.2 ± 1.5%. By combining VL3H and VL3P, the resulting PONDR VL3E achieved an accuracy of 86.7 ± 1.4%. This is a significant improvement over our previous PONDRs VLXT (71.6 ± 1.3%) and VL2 (80.9 ± 1.4%). The new disorder predictors with the corresponding datasets are freely accessible through the web server at .
ATP stimulates GRK-3 phosphorylation and β-arrestin-2-dependent internalization of P2X7 receptor
Authors: Feng Ying-Hong , Wang Liqin , Wang Qifang , Li Xin , Zeng Robin , Gorodeski George I.
Published: February 2005

The objective of this study was to understand the mechanisms involved in P2X7 receptor activation. Treatments with ATP or with the P2X7 receptor-specific ligand 2′,3′- O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) induced pore formation, but the effect was slower in CaSki cells expressing endogenous P2X7 receptor than in human embryonic kidney (HEK)-293 cells expressing exogenous P2X7 receptor (HEK-293-hP2X7-R). In both types of cells Western blots revealed expression of three forms of the receptor: the functional 85-kDa form present mainly in the membrane and 65- and 18-kDa forms expressed in both the plasma membrane and the cytosol. Treatments with ATP transiently decreased the 85-kDa form and increased the 18-kDa form in the membrane, suggesting internalization, degradation, and recycling of the receptor. In CaSki cells ATP stimulated phosphorylation of the 85-kDa form on tyrosine and serine residues. Phosphorylation on threonine residues increased with added ATP, and it increased ATP requirements for phosphorylation on tyrosine and serine residues, suggesting a dominant-negative effect. In both CaSki and in HEK-293-hP2X7-R cells ATP also increased binding of the 85-kDa form to G protein-coupled receptor kinase (GRK)-3, β-arrestin-2, and dynamin, and it stimulated β-arrestin-2 redistribution into submembranous regions of the cell. These results suggest a novel mechanism for P2X7 receptor action, whereby activation involves a GRK-3-, β-arrestin-2-, and dynamin-dependent internalization of the receptor into clathrin domains, followed in part by receptor degradation as well as receptor recycling into the plasma membrane.
Influence of increased mechanical loading by hypergravity on the microtubule cytoskeleton and prostaglandin E2 release in primary osteoblasts
Authors: Searby Nancy D. , Steele Charles R. , Globus Ruth K.
Published: February 2005

Cells respond to a wide range of mechanical stimuli such as fluid shear and strain, although the contribution of gravity to cell structure and function is not understood. We hypothesized that bone-forming osteoblasts are sensitive to increased mechanical loading by hypergravity. A centrifuge suitable for cell culture was developed and validated, and then primary cultures of fetal rat calvarial osteoblasts at various stages of differentiation were mechanically loaded using hypergravity. We measured microtubule network morphology as well as release of the paracrine factor prostaglandin E2 (PGE2). In immature osteoblasts, a stimulus of 10× gravity (10 g) for 3 h increased PGE2 2.5-fold and decreased microtubule network height 1.12-fold without affecting cell viability. Hypergravity (3 h) caused dose-dependent (5–50 g) increases in PGE2 (5.3-fold at 50 g) and decreases (1.26-fold at 50 g) in microtubule network height. PGE2 release depended on duration but not orientation of the hypergravity load. As osteoblasts differentiated, sensitivity to hypergravity declined. We conclude that primary osteoblasts demonstrate dose- and duration-dependent sensitivity to gravitational loading, which appears to be blunted in mature osteoblasts.
Matrix metalloproteinases mediate β-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes
Authors: Menon Bindu , Singh Mahipal , Singh Krishna
Published: February 2005

Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in β-adrenergic receptor (β-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). β-AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed β-AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited β-AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with β1-integrins after β-AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of β1-integrin signaling using laminin inhibited the increased association of MMP-2 with β1-integrins. β-AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) β-AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits β-AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with β1 integrins and poly-ADP-ribose-polymerase cleavage.
Furosemide and Mannitol Suppression of Epileptic Activity in the Human Brain
Authors: Haglund Michael M. , Hochman Daryl W.
Published: February 2005

Most research on basic mechanisms of epilepsy and the design of new antiepileptic drugs has focused on synaptic transmission or action potential generation. However, a number of laboratory studies have suggested that nonsynaptic mechanisms, such as modulation of electric field interactions via the extracellular space (ECS), might also contribute to neuronal hypersynchrony and epileptogenicity. To date, a role for nonsynaptic modulation of epileptic activity in the human brain has not been investigated. Here we studied the effects of molecules that modulate the volume and water content of the ECS on epileptic activity in patients suffering from neocortical and mesial temporal lobe epilepsy. Electrophysiological and optical imaging data were acquired from the exposed cortices of anesthetized patients undergoing surgical treatment for intractable epilepsy. Patients were given a single intravenous injection containing either 20 mg furosemide (a cation-chloride cotransporter antagonist) or 50 g mannitol (an osmolyte). Furosemide and mannitol both significantly suppressed spontaneous epileptic spikes and electrical stimulation-evoked epileptiform discharges in all subjects, completely blocking all epileptic activity in some patients without suppressing normal electroencephalographic activity. Optical imaging suggested that the spread of electrical stimulation-evoked activity over the cortex was significantly reduced by these treatments, but the magnitude of neuronal activation near the stimulating electrode was not diminished. These results suggest that nonsynaptic mechanisms play a critical role in modulating the epileptogenicity of the human brain. Furosemide and other drugs that modulate the ECS might possess clinically useful antiepileptic properties, while avoiding the side effects associated with the suppression of neuronal excitability.
Prehension Synergies: Trial-to-Trial Variability and Principle of Superposition During Static Prehension in Three Dimensions
Authors: Shim Jae Kun , Latash Mark L. , Zatsiorsky Vladimir M.
Published: February 2005

We performed three-dimensional analysis of the conjoint changes of digit forces during prehension (prehension synergies) and tested applicability of the principle of superposition to three-dimensional tasks. Subjects performed 25 trials at statically holding a handle instrumented with six-component force/moment sensors under seven external torque conditions; –0.70, –0.47, –0.23, 0.00, 0.23, 0.47, and 0.70 Nm about a horizontal axis in the plane passing through the centers of all five digit force sensors (the grasp plane). The total weight of the system was always 10.24 N. The trial-to-trial variability of the forces produced by the thumb and the virtual finger (an imagined finger producing the same mechanical effects as all 4 finger forces and moments combined) increased in all three dimensions with the external torque magnitude. The sets of force and moment variables associated with the moment production about the vertical axis in the grasp plane and the axis orthogonal to the grasp plane consisted of two noncorrelated subsets each; one subset of variables was related to the control of grasping forces ( grasp control) and the other sassociated with the control of the orientation of the hand-held object ( torque control). The variables associated with the moment production about the horizontal axis in the grasp plane did not include the grip force (the normal thumb and virtual finger forces) and showed more complex noncorrelated subsets. We conclude that the principle of superposition is valid for the prehension in three dimensions. The observed high correlations among forces and moments associated with the control of object orientation could be explained by chain effects, the sequences of cause-effect relations necessitated by mechanical constraints.
Transient Role for CD1d-Restricted Natural Killer T Cells in the Formation of Atherosclerotic Lesions
Authors: Aslanian Ara M. , Chapman Harold A. , Charo Israel F.
Published: February 2005

Objective—CD1d-restricted natural killer T (NKT) cells are reported to play a proatherogenic role in the development of atherosclerosis. However, the contribution of NKT cells to mature lesion formation and the effector mechanisms through which they act are unknown.

Methods and Results—We measured lesion size in CD1d-null (CD1d−/−) mice on the low-density lipoprotein (LDL) receptor–deficient (LDLR−/−) genetic background after 4, 8, and 12 weeks of feeding on a Western diet. Lesions inCD1d−/−LDLR−/−mice were 47% smaller at 4 weeks thanCD1d+/+LDLR−/−controls; however, there were no differences in lesion size betweenCD1d−/−LDLR−/−andCD1d+/+LDLR−/−mice at 8 or 12 weeks. We found that although NKT cells were present in the aortic arch ofCD1d+/+LDLR−/−mice on the Western diet, no differences in mRNA abundance for Th1 or Th2 cytokines were observed betweenCD1d−/−LDLR−/−andCD1d+/+LDLR−/−mice.

Conclusions—CD1d-restricted NKT cells contribute to the formation of fatty streaks; however, their influence on lesion progression is transient, and they do not significantly affect the inflammatory cytokine milieu of mature lesions.
Taking the Reasons for Human Rights Seriously
Authors: Ci Jiwei
Published: February 2005

The human rights discourse is vitiated by its tendency to reification, a tendency manifest in an ideologically motivated failure to take the reasons for human rights seriously. When a set of rights fall short, in range or strength, of the reasons adduced for them, any claim to the universality and priority of the rights in question is open to the charge of falsification and reification. Such a claim invites immanent critique insofar as a human rights discourse fails to take its own reasons seriously by working out a set of rights commensurate with them. Further critique is necessary if the human rights concept as such can be shown to be incapable of living up to the best reasons for human rights, in the shape, the author argues, of agency-based reasons. These kinds of critique, especially the latter, can serve as an antidote to the reifying tendency of the human rights discourse.