bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein Kinase B/Akt Is Present in Activated Form throughout the Entire Replicative Cycle of ΔUS3 Mutant Virus but Only at Early Times after Infection with Wild-Type Herpes Simplex Virus 1
Authors: Benetti Luca , Roizman Bernard
Published: March 2006

The product of the herpes simplex virus 1 (HSV-1) US3 gene is a multifunctional serine-threonine protein kinase that can block apoptosis induced by proapoptotic cellular proteins, exogenous agents, or replication-defective viruses. Earlier studies showed that the US3 kinase activates and functionally overlaps cellular protein kinase A (PKA). In this study we examined the status of phosphatidylinositol 3-kinase [PI(3)K] and of its effector, protein kinase B/Akt (PKB/Akt), a component of a major pathway of mammalian antiapoptotic signaling systems. We report the following. (i) Infection of target cells with HSV-1 induces transient phosphorylation of serine 473 of PKB/Akt early in infection, with a mechanism that is dependent on PI(3)K. Inhibition of PI(3)K induced apoptosis in mock-infected or ΔUS3 mutant-virus-infected but not in wild-type-virus-infected cells and reduced the accumulation of specific viral gene products, including the US3 protein kinase, but had a marginal effect on virus yields. (ii) At later times after infection, the total amounts of PKB/Akt decreased and phosphorylated PKB/Akt forms disappeared in a US3-dependent and protein phosphatase 2A-independent manner. (iii) Activation of PKA by forskolin did not mediate significant dephosphorylation of PKB/Akt. Our results are consistent with the model that PKB/Akt is activated early in infection and acts to block apoptosis in infected cells prior to the accumulation of US3 protein kinase and that it persists and continues to function as an antiapoptotic protein in the absence of US3 but becomes redundant or even inimical once US3 protein kinase accumulates in effective amounts.
State and Role of Src Family Kinases in Replication of Herpes Simplex Virus 1
Authors: Liang Yu , Roizman Bernard
Published: March 2006

An earlier report showed that infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) interacts with the SH3 domains of a recently discovered adaptor protein, CIN85. Here, we report the following. (i) ICP0 also interacts with other SH3 domain-containing proteins and, in particular, with nonneuronal members of the Src kinase family. (ii) HSV-1 infection enhanced the activating phosphorylation of Tyr416 of the members of the Src kinase family, modestly enhanced the kinase activity of Src, and posttranslationally modified at least one additional member of the Src kinase family by phosphorylation in a manner dependent on the viral gene products ICP0, unique short 3 (U
S
3), and unique long 13 (U
L
13). (iii) To define the roles of Src kinase family members, we examined the accumulation of viral proteins, DNA, and mRNA and virus yields from wild-type mouse embryo fibroblasts and sibling cells lacking Src, Fyn, and Yes (SYF
−
); a mutant cell line, +Src, in which Src was restored to SYF
−
cells; and the mutant cell line (CSK
−
) lacking the negative regulator Csk gene of the Src kinase family. Representative α, β, and γ
2
proteins accumulated in the largest amounts in SYF
−
cells and the smallest amounts in +Src compared to wild-type cells. The CSK
−
cells yielded smaller amounts of the γ
2
protein and at least 10-fold less virus than wild-type cells. We conclude that HSV-1 proteins regulate the activities of Src family kinases to achieve optimal viral yields in the course of viral replication.
ICP0 Is Not Required for Efficient Stress-Induced Reactivation of Herpes Simplex Virus Type 1 from Cultured Quiescently Infected Neuronal Cells
Authors: Miller Craig S. , Danaher Robert J. , Jacob Robert J.
Published: March 2006

Viral genes sufficient and required for herpes simplex virus type 1 (HSV-1) reactivation were identified using neuronally differentiated PC12 cells (ND-PC12 cells) in which quiescent infections with wild-type and recombinant strains were established. In this model, the expression of ICP0, VP16, and ICP4 from adenovirus vectors was sufficient to reactivate strains 17
+
and KOS. The transactivators induced similar levels of reactivation with KOS; however, 17
+
responded more efficiently to ICP0. To identify viral transactivators required for reactivation, we examined quiescently infected PC12 cell cultures (QIF-PC12 cell cultures) established with HSV-1 deletion mutants R7910 (ΔICP0), KD6 (ΔICP4), and
in
1814, a virus containing an insertion mutation in VP16. Although growth of these mutant viruses was impaired in ND-PC12 cells, R7910 and
in
1814 reactivated at levels equivalent to or better than their respective parental controls following stress (i.e., heat or forskolin) treatment. After treatment with trichostatin A,
in
1814 and 17
+
reactivated efficiently, whereas the F strain and R7910 reactivated inefficiently. In contrast, KD6 failed to reactivate. In experiments with the recombinant KM100, which contains the
in
1814 mutation in VP16 and the n212 mutation in ICP0, spontaneous and stress-induced reactivation was observed. However, two strains, V422 and KM110, which lack the acidic activation domain of VP16, did not reactivate above low spontaneous levels after stress. These results demonstrate that in QIF-PC12 cells ICP0 is not required for efficient reactivation of HSV-1, the acidic activation domain of VP16 is essential for stress-induced HSV-1 reactivation, and HSV-1 reactivation is modulated uniquely by different treatment constraints and phenotypes.
A Putative Thiamine Transport Protein Is a Receptor for Feline Leukemia Virus Subgroup A
Authors: Mendoza Ramon , Anderson Maria M. , Overbaugh Julie
Published: March 2006

Feline leukemia virus (FeLV) is a horizontally transmitted virus that causes a variety of proliferative and immunosuppressive diseases in cats. There are four subgroups of FeLV, A, B, C, and T, each of which has a distinct receptor requirement. The receptors for all but the FeLV-A subgroup have been defined previously. Here, we report the identification of the cellular receptor for FeLV-A, which is the most transmissible form of FeLV. The receptor cDNA was isolated using a gene transfer approach, which involved introducing sequences from a feline cell line permissive to FeLV-A into a murine cell line that was not permissive. The feline cDNA identified by this method was approximately 3.5 kb, and included an open reading frame predicted to encode a protein of 490 amino acids. This feline cDNA conferred susceptibility to FeLV-A when reintroduced into nonpermissive cells, but it did not render these cells permissive to any other FeLV subgroup. Moreover, these cells specifically bound FeLV-A-pseudotyped virus particles, indicating that the cDNA encodes a binding receptor for FeLV-A. The feline cDNA shares ∼93% amino acid sequence identity with the human thiamine transport protein 1 (THTR1). The human THTR1 receptor was also functional as a receptor for FeLV-A, albeit with reduced efficiency compared to the feline orthologue. On the basis of these data, which strongly suggest the feline protein is the orthologue of human THTR1, we have named the feline receptor feTHTR1. Identification of this receptor will allow more detailed studies of the early events in FeLV transmission and may provide insights into FeLV pathogenesis.
Differential Utilization of CD134 as a Functional Receptor by Diverse Strains of Feline Immunodeficiency Virus
Authors: Willett Brian J. , McMonagle Elizabeth L. , Ridha Susan , Hosie Margaret J.
Published: March 2006

The feline homologue of CD134 (fCD134) is the primary binding receptor for feline immunodeficiency virus (FIV), targeting the virus preferentially to activated CD4
+
helper T cells. However, with disease progression, the cell tropism of FIV broadens such that B cells and monocytes/macrophages become significant reservoirs of proviral DNA, suggesting that receptor utilization may alter with disease progression. We examined the receptor utilization of diverse strains of FIV and found that all strains tested utilized CD134 as the primary receptor. Using chimeric feline × human CD134 receptors, the primary determinant of receptor function was mapped to the first cysteine-rich domain (CRD1) of fCD134. For the PPR and B2542 strains, the replacement of CDR1 of fCD134 (amino acids 1 to 64) with human CD134 (hCD134) alone was sufficient to confer nearly optimal receptor function. However, evidence of differential utilization of CD134 was revealed, since strains GL8, CPGammer (CPG41), TM2, 0827, and NCSU1 required determinants in the region spanning amino acids 65 to 85, indicating that these strains may require a more stringent interaction for infection to proceed.
Effects of Type I Interferons on Friend Retrovirus Infection
Authors: Gerlach Nicole , Schimmer Simone , Weiss Siegfried , Kalinke Ulrich , Dittmer Ulf
Published: March 2006

The type I interferon (IFN) response plays an important role in the control of many viral infections. However, since there is no rodent animal model for human immunodeficiency virus, the antiviral effect of IFN-α and IFN-β in retroviral infections is not well characterized. In the current study we have used the Friend virus (FV) model to determine the activity of type I interferons against a murine retrovirus. After FV infection of mice, IFN-α and IFN-β could be measured between 12 and 48 h in the serum. The important role of type I IFN in the early immune defense against FV became evident when mice deficient in IFN type I receptor (IFNAR
−/−
) or IFN-β (IFN-β
−/−
) were infected. The levels of FV infection in plasma and in spleen were higher in both strains of knockout mice than in C57BL/6 wild-type mice. This difference was induced by an antiviral effect of IFN-α and IFN-β and was most likely mediated by antiviral enzymes as well as by an effect of these IFNs on T-cell responses. Interestingly, the lack of IFNAR and IFN-β enhanced viral loads during acute and chronic FV infection. Exogenous IFN-α could be used therapeutically to reduce FV replication during acute but not chronic infection. These findings indicate that type I IFN plays an important role in the immediate antiviral defense against Friend retrovirus infection.
Downregulation of Varicella-Zoster Virus (VZV) Immediate-Early ORF62 Transcription by VZV ORF63 Correlates with Virus Replication In Vitro and with Latency
Authors: Hoover Susan E. , Cohrs Randall J. , Rangel Zoila G. , Gilden Donald H. , Munson Peter , Cohen Jeffrey I.
Published: March 2006

Varicella-zoster virus (VZV) open reading frame 63 (ORF63) protein is expressed during latency in human sensory ganglia. Deletion of ORF63 impairs virus replication in cell culture and establishment of latency in cotton rats. We found that cells infected with a VZV ORF63 deletion mutant yielded low titers of cell-free virus and produced very few enveloped virions detectable by electron microscopy compared with those infected with parental virus. Microarray analysis of cells infected with a recombinant adenovirus expressing ORF63 showed that transcription of few human genes was affected by ORF63; a heat shock 70-kDa protein gene was downregulated, and several histone genes were upregulated. In experiments using VZV transcription arrays, deletion of ORF63 from VZV resulted in a fourfold increase in expression of ORF62, the major viral transcriptional activator. A threefold increase in ORF62 protein was observed in cells infected with the ORF63 deletion mutant compared with those infected with parental virus. Cells infected with ORF63 mutants impaired for replication and latency (J. I. Cohen, T. Krogmann, S. Bontems, C. Sadzot-Delvaux, and L. Pesnicak, J. Virol.79:5069-5077, 2005) showed an increase in ORF62 transcription compared with those infected with parental virus. In contrast, cells infected with an ORF63 mutant that is not impaired for replication or latency showed ORF62 RNA levels equivalent to those in cells infected with parental virus. The ability of ORF63 to downregulate ORF62 transcription may play an important role in virus replication and latency.
Human T-Lymphotropic Virus Type 1 Mitochondrion-Localizing Protein p13
II
Is Required for Viral Infectivity In Vivo
Authors: Hiraragi Hajime , Kim Seung-Jae , Phipps Andrew J. , Silic-Benussi Micol , Ciminale Vincenzo , Ratner Lee , Green Patrick L. , Lairmore Michael D.
Published: March 2006

Human T-lymphotropic virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia, encodes unique regulatory and accessory proteins in the pX region of the provirus, including the open reading frame II product p13
II
. p13
II
localizes to mitochondria, binds farnesyl pyrophosphate synthetase, an enzyme involved in posttranslational farnesylation of Ras, and alters Ras-dependent cell signaling and control of apoptosis. The role of p13
II
in virus infection in vivo remains undetermined. Herein, we analyzed the functional significance of p13
II
in HTLV-1 infection. We compared the infectivity of a human B-cell line that harbors an infectious molecular clone of HTLV-1 with a selective mutation that prevents the translation of p13
II
(729.ACH.p13) to the infectivity of a wild-type HTLV-1-expressing cell line (729.ACH). 729.ACH and 729.ACH.p13 producer lines had comparable infectivities for cultured rabbit peripheral blood mononuclear cells (PBMC), and the fidelity of the start codon mutation in ACH.p13 was maintained after PBMC passage. In contrast, zero of six rabbits inoculated with 729.ACH.p13 cells failed to establish viral infection, whereas six of six rabbits inoculated with wild-type HTLV-1-expressing cells (729.ACH) were infected as measured by antibody responses, proviral load, and HTLV-1 p19 matrix antigen production from ex vivo-cultured PBMC. Our data are the first to indicate that the HTLV-1 mitochondrion-localizing protein p13
II
has an essential biological role during the early phase of virus infection in vivo.
Measles Virus Targets DC-SIGN To Enhance Dendritic Cell Infection
Authors: de Witte Lot , Abt Marion , Schneider-Schaulies Sibylle , van Kooyk Yvette , Geijtenbeek Teunis B. H.
Published: March 2006

Dendritic cells (DCs) are involved in the pathogenesis of measles virus (MV) infection by inducing immune suppression and possibly spreading the virus from the respiratory tract to lymphatic tissues. It is becoming evident that DC function can be modulated by the involvement of different receptors in pathogen interaction. Therefore, we have investigated the relative contributions of different MV-specific receptors on DCs to MV uptake into and infection of these cells. DCs express the MV receptors CD46 and CD150, and we demonstrate that the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is a novel receptor for laboratory-adapted and wild-type MV strains. The ligands for DC-SIGN are both MV glycoproteins F and H. In contrast to CD46 and CD150, DC-SIGN does not support MV entry, since DC-SIGN does not confer susceptibility when stably expressed in CHO cells. However, DC-SIGN is important for the infection of immature DCs with MV, since both attachment and infection of immature DCs with MV are blocked in the presence of DC-SIGN inhibitors. Our data demonstrate that DC-SIGN is crucial as an attachment receptor to enhance CD46/CD150-mediated infection of DCs in
cis
. Moreover, MV might not only target DC-SIGN to infect DCs but may also use DC-SIGN for viral transmission and immune suppression.
Porcine Circovirus 2 Uses Heparan Sulfate and Chondroitin Sulfate B Glycosaminoglycans as Receptors for Its Attachment to Host Cells
Authors: Misinzo Gerald , Delputte Peter L. , Meerts Peter , Lefebvre David J. , Nauwynck Hans J.
Published: March 2006

Monocyte/macrophage lineage cells are target cells in vivo for porcine circovirus 2 (PCV2) replication. The porcine monocytic cell line 3D4/31 supports PCV2 replication in vitro, and attachment and internalization kinetics of PCV2 have been established in these cells. However, PCV2 receptors remain unknown. Glycosaminoglycans (GAG) are used by several viruses as receptors. The present study examined the role of GAG in attachment and infection of PCV2. Heparin, heparan sulfate (HS), chondroitin sulfate B (CS-B), but not CS-A, and keratan sulfate reduced PCV2 infection when these GAG were incubated with PCV2 prior to and during inoculation of 3D4/31 cells. Enzymatic removal of HS and CS-B prior to PCV2 inoculation of 3D4/31 cells significantly reduced PCV2 infection. Similarly, when PCV2 virus-like particles (VLP) were allowed to bind onto 3D4/31 cells in the presence of heparin and CS-B, attachment was strongly reduced. Titration of field isolates and low- and high-passage laboratory strains of PCV2 in the presence of heparin significantly reduced PCV2 titers, showing that the capacity of PCV2 to bind GAG was not acquired during in vitro cultivation but is an intrinsic feature of wild-type virus. When Chinese hamster ovary (CHO) cells were inoculated with PCV2, relative percentages of PCV2-infected cells were 27% ± 8% for HS-deficient and 12% ± 10% for GAG-deficient cells compared to wild-type cells (100%). Furthermore, it was shown using heparin-Sepharose chromatography that both PCV2 and PCV2 VLP directly interacted with heparin. Together, these results show that HS and CS-B are attachment receptors for PCV2.
Expression of the Interleukin-7 Receptor Alpha Chain (CD127) on Virus-Specific CD8
+
T Cells Identifies Functionally and Phenotypically Defined Memory T Cells during Acute Resolving Hepatitis B Virus Infection
Authors: Boettler Tobias , Panther Elisabeth , Bengsch Bertram , Nazarova Natalja , Spangenberg Hans Christian , Blum Hubert E. , Thimme Robert
Published: March 2006

Virus-specific CD8
+
T cells play a central role in the outcome of several viral infections, including hepatitis B virus (HBV) infection. A key feature of virus-specific CD8
+
T cells is the development of memory. The mechanisms resulting in the establishment of T-cell memory are still only poorly understood. It has been suggested that T-cell memory may depend on the survival of virus-specific CD8
+
T cells in the contraction phase. Indeed, a population of effector cells that express high levels of the interleukin-7 receptor alpha chain (CD127) as the precursors of memory CD8
+
T cells has recently been identified in mice. However, very little information is currently available about the kinetics of CD127 expression in an acute resolving viral infection in humans and its association with disease pathogenesis, viral load, and functional and phenotypical T-cell characteristics. To address these important issues, we analyzed the HBV-specific CD8
+
T-cell response longitudinally in a cohort of six patients with acute HBV infection who spontaneously cleared the virus. We observed the emergence of CD127 expression on antigen-specific CD8
+
memory T cells during the course of infection. Importantly, the up-regulation of CD127 correlated phenotypically with a loss of CD38 and PD-1 expression and acquisition of CCR7 expression: functionally with an enhanced proliferative capacity and clinically with the decline in serum alanine aminotransferase levels and viral clearance. These results suggest that the expression of CD127 is a marker for the development of functionally and phenotypically defined antigen-specific CD8
+
memory T cells in cleared human viral infections.
UL26-Deficient Human Cytomegalovirus Produces Virions with Hypophosphorylated pp28 Tegument Protein That Is Unstable within Newly Infected Cells
Authors: Munger Joshua , Yu Dong , Shenk Thomas
Published: March 2006

The human cytomegalovirus UL26 open reading frame encodes proteins of 21 and 27 kDa that result from the use of two different in-frame initiation codons. The UL26 protein is a constituent of the virion and thus is delivered to cells upon viral entry. We have characterized a mutant of human cytomegalovirus in which the UL26 open reading frame has been deleted. The UL26 deletion mutant has a profound growth defect, the magnitude of which is dependent on the multiplicity of infection. Two very early defects were discovered. First, even though they were present in normal amounts within mutant virions, the UL99-coded pp28 and UL83-coded pp65 tegument proteins were present in reduced amounts at the earliest times assayed within newly infected cells; second, there was a delay in immediate-early mRNA and protein accumulation. Further analysis revealed that although wild-type levels of the pp28 tegument protein were present in UL26 deletion mutant virions, the protein was hypophosphorylated. We conclude that the UL26 protein influences the normal phosphorylation of at least pp28 in virions and possibly additional tegument proteins. We propose that the hypophosphorylation of tegument proteins causes their destabilization within newly infected cells, perhaps disrupting the normal detegumentation process and leading to a delay in the onset of immediate-early gene expression.
ICP27 Interacts with the C-Terminal Domain of RNA Polymerase II and Facilitates Its Recruitment to Herpes Simplex Virus 1 Transcription Sites, Where It Undergoes Proteasomal Degradation during Infection
Authors: Dai-Ju Jenny Q. , Li Ling , Johnson Lisa A. , Sandri-Goldin Rozanne M.
Published: March 2006

Herpes simplex virus 1 (HSV-1) ICP27 has been shown to interact with RNA polymerase II (RNAP II) holoenzyme. Here, we show that ICP27 interacts with the C-terminal domain (CTD) of RNAP II and that ICP27 mutants that cannot interact fail to relocalize RNAP II to viral transcription sites, suggesting a role for ICP27 in RNAP II recruitment. Using monoclonal antibodies specific for different phosphorylated forms of the RNAP II CTD, we found that the serine-2 phosphorylated form, which is found predominantly in elongating complexes, was not recruited to viral transcription sites. Further, there was an overall reduction in phosphoserine-2 staining. Western blot analysis revealed that there was a pronounced decrease in the phosphoserine-2 form and in overall RNAP II levels in lysates from cells infected with wild-type HSV-1. There was no appreciable difference in cdk9 levels, suggesting that protein degradation rather than dephosphorylation was occurring. Treatment of infected cells with proteasome inhibitors MG-132 and lactacystin prevented the decrease in the phosphoserine-2 form and in overall RNAP II levels; however, there was a concomitant decrease in the levels of several HSV-1 late proteins and in virus yield. Proteasomal degradation has been shown to resolve stalled RNAP II complexes at sites of DNA damage to allow 3′ processing of transcripts. Thus, we propose that at later times of infection when robust transcription and DNA replication are occurring, elongating complexes may collide and proteasomal degradation may be required for resolution.
Fitness Cost of Escape Mutations in p24 Gag in Association with Control of Human Immunodeficiency Virus Type 1
Authors: Martinez-Picado Javier , Prado Julia G. , Fry Elizabeth E. , Pfafferott Katja , Leslie Alasdair , Chetty Senica , Thobakgale Christina , Honeyborne Isobel , Crawford Hayley , Matthews Philippa , Pillay Tilly , Rousseau Christine , Mullins James I. , Brander Christian , Walker Bruce D. , Stuart David I. , Kiepiela Photini , Goulder Philip
Published: March 2006

Mutational escape by human immunodeficiency virus (HIV) from cytotoxic T-lymphocyte (CTL) recognition is a major challenge for vaccine design. However, recent studies suggest that CTL escape may carry a sufficient cost to viral replicative capacity to facilitate subsequent immune control of a now attenuated virus. In order to examine how limitations can be imposed on viral escape, the epitope TSTLQEQIGW (TW10 [Gag residues 240 to 249]), presented by two HLA alleles associated with effective control of HIV, HLA-B*57 and -B*5801, was investigated. The in vitro experiments described here demonstrate that the dominant TW10 escape mutation, T242N, reduces viral replicative capacity. Structural analysis reveals that T242 plays a critical role in defining the start point and in stabilizing helix 6 within p24 Gag, ensuring that escape occurs at a significant cost. A very similar role is played by Thr-180, which is also an escape residue, but within a second p24 Gag epitope associated with immune control. Analysis of HIV type 1
gag
in 206 B*57/5801-positive subjects reveals three principle alternative TW10-associated variants, and each is strongly linked to concomitant additional variants within p24 Gag, suggesting that functional constraints operate against their occurrence alone. The extreme conservation of p24 Gag and the predictable nature of escape variation resulting from these tight functional constraints indicate that p24 Gag may be a critical immunogen in vaccine design and suggest novel vaccination strategies to limit viral escape options from such epitopes.
A Versatile Transreplication-Based System To Identify Cellular Proteins Involved in Geminivirus Replication
Authors: Morilla Gabriel , Castillo Araceli G. , Preiss Werner , Jeske Holger , Bejarano Eduardo R.
Published: March 2006

A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus
Tomato yellow leaf curl Sardinia virus
(TYLCSV) is described. Transgenic
Nicotiana benthamiana
plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal
trans
replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing.
Infection and Persistence of Rhesus Monkey Rhadinovirus in Immortalized B-Cell Lines
Authors: Bilello John P. , Lang Sabine M. , Wang Fred , Aster Jon C. , Desrosiers Ronald C.
Published: March 2006

Similar to its close relative human herpesvirus 8, rhesus monkey rhadinovirus (RRV) persists predominantly in B cells of its natural host. Rhesus monkey B-cell lines immortalized by the Epstein-Barr-related virus from rhesus monkeys (rhEBV) were used as targets for infection by RRV. These cultured B cells were susceptible to infection by RRV and continued to produce low titers of RRV for months of continuous culture. Infection by RRV did not detectably alter the growth rates of these B-cell lines when it was measured at standard or reduced serum concentrations. Depending on the cell line, 5 to 40% of the B cells stained positive for the RRV genome by fluorescence in situ hybridization (FISH). Most RRV-positive cells showed a fine punctate nuclear staining pattern consistent with latent infection, while a small minority of cells (0.2 to 1%) contained large, intensely staining nuclear foci consistent with productive, replicative infection. Greater than 90% of the cells were rhEBV genome positive in a pattern consistent with latent infection, and again only a small minority of cells showed a productive, replicative staining pattern. Dual, two-color FISH staining revealed coinfection of numerous cells with both RRV and rhEBV, but productive replication of RRV and rhEBV was always observed in separate cells, never in the same cell. Thus, productive replication of RRV is unlinked to that of rhEBV; factors that influence activation to productive replication act separately on RRV and rhEBV, even within the same cell. The percentage of B cells expressing green fluorescent protein (GFP) early after infection with a recombinant RRV containing a GFP reporter gene was dose dependent and at a low multiplicity of infection increased progressively over time until 14 to 17 days after infection. These results establish a naturalistic cell culture system for the study of infection and persistence by RRV in rhesus monkey B cells.
Brd4 Is Involved in Multiple Processes of the Bovine Papillomavirus Type 1 Life Cycle
Authors: Ilves Ivar , Mäemets Kristina , Silla Toomas , Janikson Kadri , Ustav Mart
Published: March 2006

Brd4 protein has been proposed to act as a cellular receptor for the bovine papillomavirus type 1 (BPV1) E2 protein in the E2-mediated chromosome attachment and mitotic segregation of viral genomes. Here, we provide data that show the involvement of Brd4 in multiple early functions of the BPV1 life cycle, suggest a Brd4-dependent mechanism for E2-dependent transcription activation, and indicate the role of Brd4 in papillomavirus and polyomavirus replication as well as cell-specific utilization of Brd4-linked features in BPV1 DNA replication. Our data also show the potential therapeutic value of the disruption of the E2-Brd4 interaction for the development of antiviral drugs.
Phylogenetic Evidence for the Rapid Evolution of Human B19 Erythrovirus
Authors: Shackelton Laura A. , Holmes Edward C.
Published: March 2006

Human B19 erythrovirus is a ubiquitous viral pathogen, commonly infecting individuals before adulthood. As with all autonomous parvoviruses, its small single-stranded DNA genome is replicated with host cell machinery. While the mechanism of parvovirus genome replication has been studied in detail, the rate at which B19 virus evolves is unknown. By inferring the phylogenetic history and evolutionary dynamics of temporally sampled B19 sequences, we observed a surprisingly high rate of evolutionary change, at approximately 10
−4
nucleotide substitutions per site per year. This rate is more typical of RNA viruses and suggests that high mutation rates are characteristic of the
Parvoviridae
.
Role of polymerase chain reaction and immunocytochemistry in the cytological assessment of lymphoid proliferations
Authors: Venkatraman L , Catherwood M A , Patterson A , Lioe T F , McCluggage W G , Anderson N H
Published: March 2006

Background:Fine-needle aspiration cytology (FNAC) is used as a screening test to evaluate lymphadenopathy. The combined use of genetic analysis and flow cytometry for immunophenotyping has increased the accuracy of diagnosis and correct categorisation of lymphomas on cytological preparations.

Aim:To show the utility of immunocytochemistry and polymerase chain reaction (PCR) in the evaluation of cytological preparations of lymph nodes.

Methods:Fine needle aspirates were obtained from 33 patients (initial presentation, n = 27; recurrence, n = 6). Routine examination was undertaken using immunocytochemistry and DNA PCR to detect clonality and specific translocations. The cytodiagnosis and subclassification of lymphoma was correlated with histological diagnosis in the available follow-up biopsies.

Results:14 patients had a cytological diagnosis of non-Hodgkin’s lymphoma (NHL), 4 had suspected NHL, 2 had atypical lymphoid proliferation and 13 had reactive hyperplasia. A World Health Organization (WHO) subtype was suggested in 8 patients. Incorporating the results of immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) gene rearrangements enabled diagnosis of lymphoma in 17 patients, including 5 of the 6 patients suspected to have NHL or an atypical lymphoid proliferation. Identification of the translocations t (14;18) and t (2;5) helped WHO categorisation in 3 of the patients. The cytological findings were confirmed in 12 out of the 13 patients for whom histological follow-up was available. Seven of the 18 lymphoma patients were managed without a subsequent biopsy. We made one false–positive diagnosis of B-cell NHL on cytology.

Conclusion:The use of immunocytochemistry and PCR is valuable in the definitive diagnosis and subtyping of malignant lymphomas on cytological preparations. The use of these techniques may avoid lymph node biopsies in some cases and allow definitive treatment based on aspirate findings alone.
Sedimentation characteristics of leucocytes can predict bacteraemia in critical care patients: Figure 1
Authors: Bogar L , Molnar Z , Kenyeres P , Tarsoly P
Published: March 2006

Background:Early detection of blood stream infection can be lifesaving, but the results of blood cultures are not usually available before 24 hours after blood sampling. An earlier indication would lead to the initiation of immediate and adequate antibiotic treatment with obvious advantages for the patient.

Objective:To evaluate the ability of leucocyte count, serum procalcitonin (PCT) concentration, erythrocyte sedimentation rate (ESR), and leucocyte antisedimentation rate (LAR) in predicting the blood culture results in critical care patients.

Methods:39 consecutive patients with their first febrile episode were investigated prospectively. LAR was determined as the percentage of leucocytes crossing the midline of a blood column upward during one hour of gravity sedimentation. The relevance of the different variables was estimated by likelihood ratio tests and area under receiver operating characteristic curves (AUC).

Results:23 patients had positive blood culture results and 16 negative. LAR was significantly higher in bacteraemic patients than in non-bacteraemic patients (p = 0.001), but leucocyte count, ESR and PCT level failed to show significant differences. Leucocyte count, PCT, and ESR yielded low discriminative values with the AUCs of 0.66, 0.64, and 0.52, respectively. LAR provided a likelihood ratio of 3.6 and an AUC of 0.80 (95% confidence interval, 0.64 to 0.95) (p = 0.002).

Conclusions:The simple LAR test can predict blood culture results and support urgent treatment decisions in critical care patients in their first febrile episode.
Carbonate, carbamate, urea, and guanidine as model species for functional groups in biological molecules  A combined density functional theory and mass spectrometry examination of polysodiation and gas-phase dissociation
Authors: Shi Tujin , Zhao Junfang , Shek PY Iris , Hopkinson Alan C , Siu KW Michael
Published: March 2006

Electrospray mass spectrometric analyses of carbonate, carbamate, urea, and guanidine in the presence of sodium ions results in polysodiated complexes of the type, [M  (n  1)H + nNa]+, where M is the species analyzed and n = 2 and (or) 3. These complexes are unusual in their high sodium contents and their attractive structures. The [M  H + 2Na]+ complexes fragment to produce [Na2NH2]+, Na+, and a product ion resulting from the loss of NH3. The [M  2H + 3Na]+ complexes dissociate to give a number of fragment ions, including [Na3O]+, [Na2OH]+, [NaOCNNa]+, [Na2NH2]+, [Na3CN2]+, and Na+, depending on the elemental composition of M. These dissociation reactions were examined both experimentally by mass spectrometry and theoretically by density functional theory. Reaction profiles as calculated at B3LYP/6-311G** for disodiated carabamate, urea, and guanidine, and for all the bases in trisodiated form are given. Both experimental and theory show [Na3CO3]+ to have high stability towards collisional activation and to preferentially dissociate to give [Na3O]+ and CO2, rather than Na+ and Na2CO3.Key words: carbonate, carbamate, urea, guanidine, electrospray, collision-induced dissociation, density functional theory, sodium ion binding energies.
Fragmentation pathways of negative ions produced by electrospray ionization of acyclic dicarboxylic acids and derivatives
Authors: Grossert J Stuart , Fancy Paul D , White Robert L
Published: March 2006

Fragmentation pathways have been studied on the monoanions formed during electrospray ionization of a wide range of aliphatic dicarboxylic acids and their monoesters. All negative ion spectra were obtained from alcoholic or aqueous methanolic solutions without buffers or adjustment of pH, using either a Finnigan LCQ ion trap or a VG-Micromass Quattro triple quadrupole mass spectrometer. Fragmentation pathways were studied using collision-induced dissociation and isotopic-labelling techniques. Two primary fragmentation pathways of the dicarboxylic acid monoanions were observed, namely decarboxylation of the non-ionized carboxyl group and loss of water from this group. The fragmentations were strongly dependent on the chain lengths of the diacids. In the case of a monoester anion, loss of a molecule of alcohol paralleled the loss of water from the diacid monoanion. Losses of water or alcohol were shown to lead to formation of reactive ynolate anions (HOOC(CH2)xC≡CO, x = 39), which in the ion trap spectrometer engaged in complex ion  molecule reactions consistent with the chemistry of these anions. For the longer chains (x > 6), the interactions between the ionized and non-ionized carboxyl groups led to readily formed ionneutral complexes, which are described as a neutral molecule (ROH, R = H or alkyl) held by a pair of molecular tweezers.Key words: ESI-MS/MS on negative ions, fragmentation pathways of acyclic carboxylic acid monoanions, ionmolecule reactions in an ion trap mass spectrometer, hydrogendeuterium exchange in a gas-phase anionneutral complex.
Combining HD exchange and ESI-FAIMS-MS for detecting gas-phase conformers of equine cytochrome c
Authors: Purves Randy W , Ells Barbara , Barnett David A , Guevremont Roger
Published: March 2006

Conformers of equine cytochrome c were investigated in the gas phase using a combination of high-field asymmetric waveform ion mobility spectrometry (FAIMS) and hydrogendeuterium (HD) exchange. Electrospray generated ions of equine cytochrome c were exposed to a low concentration of D2O vapour while being transported by a flow of nitrogen through a FAIMS device. During this transport period of about 250 ms in the FAIMS analyzer, the various conformers of multiply charged ions of cytochrome c were simultaneously undergoing HD exchange and being separated from each other. The extent of HD exchange was calculated from the observed m/z of conformers after exposure to D2O vapour in FAIMS. The complementary nature of these two methods resulted in observations supporting a greater number of conformers (e.g., at least 11 conformers were identified for the +16 charge state) than would be expected by analyzing the FAIMS data and the HD exchange data independently.Key words: FAIMS, HD exchange, mass spectrometry, protein conformations, electrospray ionization.
Thermodynamics of electrolytes in mixed solvent media  Application of the Pitzer ion interaction approach
Authors: Das Bijan
Published: March 2006

Although for a long time considerable attention has been paid to the application of the Pitzer ion interaction approach to describe the thermodynamic properties of a variety of aqueous electrolyte solutions, such studies in mixed solvent media are still scarce. The present paper discusses the application of the Pitzer ion interaction approach in the case of electrolyte solutions in mixed solvent media. In particular, a comprehensive equation for the thermodynamic properties of hydrochloric acid in dioxanewater mixtures within the temperature range 273.15 to 323.15 K has been generated in the ion interaction (Pitzer) equation form on the basis of electrochemical cell measurements of the hydrochloric acid available in the literature. It was necessary to set varying values of the exponential coefficients α1 and α2 of the standard Pitzer model in different mixed solvent media to achieve a satisfactory fit. The present model quantitatively reproduces the experimental cell electromotive forces over the entire concentration and temperature range used for the experiments. The Pitzer ion interaction approach is, thus, proved to be a very valuable method for the correlation and prediction of thermodynamic properties of electrolytes in mixed solvent media. The implications of the Pitzer ion interaction parameters (β(0), β(1) β(2), and Cϕ) have also been discussed in terms of interionic forces. The present study also stresses the need for additional emf measurements to help interpolation through the dioxane composition. Key words: Pitzer's equations, Pitzer ion interaction parameter, activity coefficients, emf, hydrochloric acid.
Fluorescence quenching of Safranine T by inorganic anions in Tween micelles
Authors: Moore Stephanie A , Moore Sarah E , Glenn Karen M , Bhattacharya Subhash C , Palepu Rama M
Published: March 2006

The fluorescence quenching of the dye Safranine T by various inorganic anions such as [Fe(CN6)]3, [Fe(CN6)]4, I, Br, NO2, and N3 in aqueous solution and in nonionic micellar solutions of Tweens (Tween 20, Tween 40, and Tween 80) was studied. The SternVolmer quenching constants and quenching rate constants were evaluated. Compared to the quenching efficiency in aqueous media, that of the micellar media was found to be lower, except when iodide ion was used as a quencher. With the exception of the iodide ion, it has been established that collisional quenching plays a major role in the photophysical process. In the case of the iodide ion, both static and collisional quenching processes play a major role, owing to the complexation of iodide ion with Tween micelles. The SternVolmer quenching constants were found to be dependent on the ionic strength of the medium.Key words: fluorescence, static quenching, Tweens, SternVolmer constant, micellar media.
Fluorinated 9-borafluorenes vs. conventional perfluoroaryl boranes  Comparative Lewis acidity
Authors: Chase Preston A , Romero Patricio E , Piers Warren E , Parvez Masood , Patrick Brian O
Published: March 2006

Perfluorinated 9-phenyl-9-borafluorene, 1, is an antiaromatic analog of the well-known tris(pentafluorophenyl)borane. Spectroscopic, structural, and electrochemical studies have been performed on 1 and its Lewis base adducts with MeCN, THF, and PMe3 with a view to assessing its comparative Lewis acid strength relative to B(C6F5)3. For the sterically undemanding Lewis base MeCN, 1 and B(C6F5)3 have comparable LA strengths, while for more sterically prominent THF, 1 is clearly the stronger Lewis acid (LA) based on competition experiments. We conclude that steric factors, rather than antiaromaticity, are the most important determinants in the LA strength differences between 1 and B(C6F5)3.Key words: boranes, Lewis acids, fluorinated compounds, heterocycles.
Glycosides from the sea cucumber Cucumaria frondosa. IV. Structure of frondosides A2-4, A2-7, and A2-8, three new minor monosulfated triterpene glycosides
Authors: Silchenko Alexandra S , Avilov Sergey A , Antonov Alexandr S , Kalinovsky Anatoly I , Dmitrenok Pavel S , Kalinin Vladimir I , Woodward Carl , Collin Peter D
Published: March 2006

Frondosides A2-4 (1), A2-7 (2), and A2-8 (3) are new monosulfated triterpene glycosides isolated from the sea cucumber Cucumaria frondosa. Their structures have been elucidated on the basis of spectral data (2D NMR and MS). Frondosides A2-7 (2) and A2-8 (3) are isomers and differ from each other only by the position of a double bond in their non-holostane-type aglycones.Key words: Cucumaria frondosa, sea cucumbers, frondosides, triterpene glycosides.
Prediction of the photoelectron spectrum for XPY2(X = H, F, Cl; Y = O, S) type of molecules by ab initio and DFT methods
Authors: Bégué Didier , Sotiropoulos Jean-marc , Pouchan Claude , Zhang Daisy Y
Published: March 2006

The present study reports the theoretical vertical ionization potentials (IPs) for all the valence electrons in six XPY2molecules by utilizing the corrected orbital energies calculated with three theoretical methods, namely, the ab initio HartreeFock (HF), and both the pure and hybrid density functional theory (DFT) methods at, respectively, the BLYP/6-311+G* and B3lYP/6-311+G* levels of theory. Evaluation of the numerical corrections to the orbital energies was achieved by comparisons with the IP values obtained via explicit computation of the energy differences between the neutral molecules and the corresponding radical cations (the ΔSCF method) and shows values from 0.9 to 1.9 eV for the HF, and positive values from 2.9 to 3.9 eV and from 1.8 to 2.4 eV for the pure and hybrid DFT methods, respectively. In contrast to the orbital energies, the ΔSCF method is shown to give consistent values among the three methods, as well as reasonable agreement with the experimental IP values.Key words: ionization potential, phosphorane, Koopmans' theorem, Janak's theorem.
Ditopic receptors of hexaamide derivatives derived from hexahomotrioxacalix[3]arene triacetic acid
Authors: Yamato Takehiko , Rahman Shofiur , Xi Zeng , Kitajima Fumika , Gil Jeong Tae
Published: March 2006

The lower rim functionalized hexahomotrioxacalix[3]arene hexaamide 7 having an amino acid moiety with cone conformation was synthesized from triol 1 by a stepwise reaction. The different extractability for alkali metal ions, transition metal ions, and alkylammonium ions from water into dichloromethane was discussed. Owing to the strong intramolecular hydrogen bond between the neighboring NH and CO groups in hexaamide 7, its affinity to metal cations was weakened. Hexaamide 7 shows a single selectivity to n-BuNH3+. The anion complexation of hexaamide 7 was also studied by 1H NMR titration experiments. Hexaamide 7 binds halides through the intermolecular hydrogen bond among the NH hydrogens of the amide in a 1:1 fashion in CDCl3. Thus, hexaamide 7 serves as a heteroditopic receptor that can complex with halides (Cl, Br) and n-BuNH3+ at the same time. The association constants calculated from the chemical shift changes of the amide protons are Ka = 536 ± 32 (mol/L)1 for Cl and Ka = 230 ± 17 (mol/L)1 for Br.Key words: hexahomotrioxacalix[3]arenes, ionophores, molecular recognition, ammonium ion, ditopic receptor.
Solubilities of H2 in H2O and D2 in D2O with dissolved boric acid and lithium hydroxide
Authors: Setthanan Uncharat , Morris David R , Lister Derek H
Published: March 2006

The solubilities of hydrogen (H2 and D2) in aqueous (H2O and D2O) solutions of lithium hydroxide or boric acid have been measured in the temperature range 2580 °C. Lithium hydroxide and boric acid cause a salting-out effect for H2 and D2. The solubilities of H2 and D2 in aqueous electrolyte solutions exhibit similar responses to temperature changes as those for pure water. The solute specific parameter (hi) for boric acid in light and heavy water is 0.07 ± 0.02 and 0.48 ± 0.26 m3 kmol1, respectively.Key words: hydrogen solubility, deuterium solubility, salting-out effect.
OPTIMAL PREEMPTIVE SEMI-ONLINE ALGORITHM FOR SCHEDULING TIGHTLY-GROUPED JOBS ON TWO UNIFORM MACHINES
Authors: JIANG YIWEI , HE YONG
Published: March 2006

In this paper, we consider a semi-online preemptive scheduling problem on two uniform machines, where we assume that all jobs have sizes between p and rp for some p > 0 and r ≥ 1. The goal is to maximize the continuous period of time (starting from time zero) when both machines are busy. We present an optimal semi-online algorithm for any combination of the job size ratio r and machine speed ratio s.