bluuebunny/crossref_metadata_2025 huggingface dataset:
High Physiological Levels of LMP1 Result in Phosphorylation of eIF2α in Epstein-Barr Virus-Infected Cells
Authors: Lam Ngan , Sandberg Mark L. , Sugden Bill
Published: January 2004

LMP1 is an Epstein-Barr virus (EBV)-encoded membrane protein essential for the proliferation of EBV-infected lymphoblasts (E. Kilger, A. Kieser, M. Baumann, and W. Hammerschmidt, EMBO J.
17:
1700-1709, 1998). LMP1 also inhibits gene expression and induces cytostasis in transfected cells when it is expressed at levels as little as twofold higher than the average for EBV-positive lymphoblasts (M. Sandberg, A. Kaykas, and B. Sugden, J. Virol.
74:
9755-9761, 2000; A. Kaykas and B. Sugden, Oncogene
19:
1400-1410, 2000). We have found that in three different clones of EBV-infected lymphoblasts the levels of expression of LMP1 in individual cells in each clone ranged over 100-fold. This difference is due to a difference in levels of the LMP1 transcript. In these clones, cells expressing high levels of LMP1 incorporated less BrdU. We also found that induction of expression of LMP1 or of a derivative of LMP1 with its transmembrane domain fused to green fluorescent protein instead of its carboxy-terminal signaling domain resulted in phosphorylation of eIF2α in EBV-negative Burkitt's lymphoma cells. This induction of phosphorylation of eIF2α was also detected in EBV-infected lymphoblasts, in which high levels of LMP1 correlated with high levels of phosphorylation of eIF2α. Our results indicate that inhibition of gene expression and of cell proliferation by LMP1 occurs normally in EBV-infected cells.
Selective Induction of Th2-Attracting Chemokines CCL17 and CCL22 in Human B Cells by Latent Membrane Protein 1 of Epstein-Barr Virus
Authors: Nakayama Takashi , Hieshima Kunio , Nagakubo Daisuke , Sato Emiko , Nakayama Masahiro , Kawa Keisei , Yoshie Osamu
Published: January 2004

Chemokines are likely to play important roles in the pathophysiology of diseases associated with Epstein-Barr virus (EBV). Here, we have analyzed the repertoire of chemokines expressed by EBV-infected B cells. EBV infection of B cells induced expression of TARC/CCL17 and MDC/CCL22, which are known to attract Th2 cells and regulatory T cells via CCR4, and also upregulated constitutive expression of MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5, which are known to attract Th1 cells and cytotoxic T cells via CCR5. Accordingly, EBV-immortalized B cells secreted these chemokines, especially CCL3, CCL4, and CCL22, in large quantities. EBV infection or stable expression of LMP1 also induced CCL17 and CCL22 in a B-cell line, BJAB. The inhibitors of the TRAF/NF-κB pathway (BAY11-7082) and the p38/ATF2 pathway (SB202190) selectively suppressed the expression of CCL17 and CCL22 in EBV-immortalized B cells and BJAB-LMP1. Consistently, transient-transfection assays using CCL22 promoter-reporter constructs demonstrated that two NF-κB sites and a single AP-1 site were involved in the activation of the CCL22 promoter by LMP1. Finally, serum CCL22 levels were significantly elevated in infectious mononucleosis. Collectively, LMP1 induces CCL17 and CCL22 in EBV-infected B cells via activation of NF-κB and probably ATF2. Production of CCL17 and CCL22, which attract Th2 and regulatory T cells, may help EBV-infected B cells evade immune surveillance by Th1 cells. However, the concomitant production of CCL3, CCL4, and CCL5 by EBV-infected B cells may eventually attract Th1 cells and cytotoxic T cells, leading to elimination of EBV-infected B cells at latency III and to selection of those with limited expression of latent genes.
The Herpes Simplex Virus ICP0 RING Finger Domain Inhibits IRF3- and IRF7-Mediated Activation of Interferon-Stimulated Genes
Authors: Lin Rongtuan , Noyce Ryan S. , Collins Susan E. , Everett Roger D. , Mossman Karen L.
Published: January 2004

Virus infection induces a rapid cellular response in cells characterized by the induction of interferon. While interferon itself does not induce an antiviral response, it activates a number of interferon-stimulated genes that collectively function to inhibit virus replication and spread. Previously, we and others reported that herpes simplex virus type 1 (HSV-1) induces an interferon -independent antiviral response in the absence of virus replication. Here, we report that the HSV-1 proteins ICP0 and vhs function in concert to disable the host antiviral response. In particular, we show that ICP0 blocks interferon regulatory factor IRF3- and IRF7-mediated activation of interferon-stimulated genes and that the RING finger domain of ICP0 is essential for this activity. Furthermore, we demonstrate that HSV-1 modifies the IRF3 pathway in a manner different from that of the small RNA viruses most commonly studied.
Human Immunodeficiency Virus Type 1 Nef Associates with Lipid Rafts To Downmodulate Cell Surface CD4 and Class I Major Histocompatibility Complex Expression and To Increase Viral Infectivity
Authors: Alexander Melissa , Bor Yeou-cherng , Ravichandran Kodimangalam S. , Hammarskjöld Marie-Louise , Rekosh David
Published: January 2004

Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.
Latent Membrane Protein 2A Inhibits Transforming Growth Factor-β1-Induced Apoptosis through the Phosphatidylinositol 3-Kinase/Akt Pathway
Authors: Fukuda Makoto , Longnecker Richard
Published: January 2004

Latent membrane protein 2A (LMP2A) blocks B-cell receptor signal transduction in vitro by binding the Syk and Lyn protein tyrosine kinases. As well as blocking B-cell signal transduction, LMP2A has been shown to activate the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, which acts as a survival signal in both B cells and epithelial cells. Transforming growth factor β1 (TGF-β1) is a multifunctional cytokine that plays important roles in regulating cell growth and differentiation in many biological systems. The loss of the growth-inhibitory response to the TGF-β1 signal is found in many cancers and is widely thought to promote tumor development. In this study, we found that LMP2A induced the phosphorylation of Akt (serine 473) in Burkitt's lymphoma cell line Ramos and in gastric carcinoma cell line HSC-39 and partially enhanced cell viability following TGF-β1 treatment. In addition, LMP2A partially inhibited TGF-β1-induced DNA fragmentation and cleavage of poly(ADP-ribose) polymerase (PARP). In the presence of LY294002, an inhibitor of PI3-K, the LMP2A-mediated inhibitory effects on TGF-β1-induced DNA fragmentation and cleavage of PARP were alleviated. Furthermore, LMP2A did not alter the levels of expression of type I and type II TGF-β1 receptors. Taken together, these results suggest that LMP2A may inhibit TGF-β1-mediated apoptosis through activation of the PI3-K/Akt pathway.
Novel Single-Cell-Level Phenotypic Assay for Residual Drug Susceptibility and Reduced Replication Capacity of Drug-Resistant Human Immunodeficiency Virus Type 1
Authors: Zhang Haili , Zhou Yan , Alcock Cecily , Kiefer Tara , Monie Daphne , Siliciano Janet , Li Quan , Pham Paul , Cofrancesco Joseph , Persaud Deborah , Siliciano Robert F.
Published: January 2004

Human immunodeficiency virus type 1 (HIV-1)-infected individuals who develop drug-resistant virus during antiretroviral therapy may derive benefit from continued treatment for two reasons. First, drug-resistant viruses can retain partial susceptibility to the drug combination. Second, therapy selects for drug-resistant viruses that may have reduced replication capacities relative to archived, drug-sensitive viruses. We developed a novel single-cell-level phenotypic assay that allows these two effects to be distinguished and compared quantitatively. Patient-derived
gag-pol
sequences were cloned into an HIV-1 reporter virus that expresses an endoplasmic reticulum-retained Env-green fluorescent protein fusion. Flow cytometric analysis of single-round infections allowed a quantitative analysis of viral replication over a 4-log dynamic range. The assay faithfully reproduced known in vivo drug interactions occurring at the level of target cells. Simultaneous analysis of single-round infections by wild-type and resistant viruses in the presence and absence of the relevant drug combination divided the benefit of continued nonsuppressive treatment into two additive components, residual virus susceptibility to the drug combination and selection for drug-resistant variants with diminished replication capacities. In some patients with drug resistance, the dominant circulating viruses retained significant susceptibility to the combination. However, in other cases, the dominant drug-resistant viruses showed no residual susceptibility to the combination but had a reduced replication capacity relative to the wild-type virus. In this case, simplification of the regimen might still allow adequate suppression of the wild-type virus. In a third pattern, the resistant viruses had no residual susceptibility to the relevant drug regimen but nevertheless had a replication capacity equivalent to that of wild-type virus. In such cases, there is no benefit to continued treatment. Thus, the ability to simultaneously analyze residual susceptibility and reduced replication capacity of drug-resistant viruses may provide a basis for rational therapeutic decisions in the setting of treatment failure.
Low Doses of Hepadnavirus Induce Infection of the Lymphatic System That Does Not Engage the Liver
Authors: Michalak Tomasz I. , Mulrooney Patricia M. , Coffin Carla S.
Published: January 2004

Woodchuck hepatitis virus (WHV), which is closely related to human hepatitis B virus and is considered to be principally hepatotropic, invades the host's lymphatic system and persists in lymphoid cells independently of whether the infection is symptomatic and serologically evident or concealed. In this study, we show, with the woodchuck model of hepatitis B, that hepadnavirus can establish an infection that engages the lymphatic system, but not the liver, and persists in the absence of virus serological markers, including antiviral antibodies. This primary occult infection is caused by wild-type virus invading the host at a quantity usually not greater than 10
3
virions. It is characterized by trace virus replication progressing in lymphatic organs and peripheral lymphoid cells that, with time, may also spread to the liver. The infection is transmissible to virus-naive hosts as an asymptomatic, indefinitely long, occult carriage of small amounts of biologically competent virus. In contrast to residual silent WHV persistence, which normally endures after the resolution of viral hepatitis and involves the liver, primary occult infection restricted to the lymphatic system does not protect against reinfection with a large, liver-pathogenic WHV dose; however, the occult infection is associated with a swift recovery from hepatitis caused by the superinfection. Our study documents that the lymphatic system is the primary target of WHV infection when small quantities of virions invade a susceptible host.
Perforin and Gamma Interferon-Mediated Control of Coronavirus Central Nervous System Infection by CD8 T Cells in the Absence of CD4 T Cells
Authors: Bergmann Cornelia C. , Parra Beatriz , Hinton David R. , Ramakrishna Chandran , Dowdell Konechi C. , Stohlman Stephen A.
Published: January 2004

Infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus produces acute and chronic demyelination. The contributions of perforin-mediated cytolysis and gamma interferon (IFN-γ) secretion by CD8+T cells to the control of infection and the induction of demyelination were examined by adoptive transfer into infected SCID recipients. Untreated SCID mice exhibited uncontrolled virus replication in all CNS cell types but had little or no demyelination. Memory CD8+T cells from syngeneic wild-type (wt), perforin-deficient, or IFN-γ-deficient (GKO) donors all trafficked into the infected CNS in the absence of CD4+T cells and localized to similar areas. Although CD8+T cells from all three donors suppressed virus replication in the CNS, GKO CD8+T cells expressed the least antiviral activity. A distinct viral antigen distribution in specific CNS cell types revealed different mechanisms of viral control. While wt CD8+T cells inhibited virus replication in all CNS cell types, cytolytic activity in the absence of IFN-γ suppressed the infection of astrocytes, but not oligodendroglia. In contrast, cells that secreted IFN-γ but lacked cytolytic activity inhibited replication in oligodendroglia, but not astrocytes. Demyelination was most severe following viral control by wt CD8+T cells but was independent of macrophage infiltration. These data demonstrate the effective control of virus replication by CD8+T cells in the absence of CD4+T cells and support the necessity for the expression of distinct effector mechanisms in the control of viral replication in distinct CNS glial cell types.
In Vivo Expansion of the Residual Tumor Antigen-Specific CD8+T Lymphocytes That Survive Negative Selection in Simian Virus 40 T-Antigen-Transgenic Mice
Authors: Schell Todd D.
Published: January 2004

Mice that express the viral oncoprotein simian virus 40 (SV40) large T antigen (T-Ag) as a transgene provide useful models for the assessment of the state of the host immune response in the face of spontaneous tumor progression. Line SV11 (H2b) mice develop rapidly progressing choroid plexus tumors due to expression of full-length T-Ag from the SV40 promoter. In addition, T-Ag expression in the thymus of SV11 mice results in the deletion of CD8+T cells specific for the three H2b-restricted immunodominant epitopes of T-Ag. Whether CD8+T cells specific for the immunorecessive H2-Db-restricted epitope V of T-Ag survive negative selection in SV11 mice has not been determined. Immunization of SV11 mice with rVV-ES-V, a recombinant vaccinia virus expressing epitope V as a minigene, resulted in the induction of weak, but reproducible, epitope V-specific cytotoxic T-lymphocyte (CTL) responses. This weak lytic response corresponded with a decreased frequency of epitope V-specific CTL that could be recruited in SV11 mice. In addition, CTL lines derived from rVV-ES-V-immunized SV11 mice had reduced avidities compared to that seen with CTL derived from healthy mice. Despite this initial weak response, significant numbers of epitope V-specific CD8+T cells were detected in SV11 mice ex vivo following a priming-boosting approach and these cells demonstrated high avidity for epitope V. The results suggest that low numbers of tumor-reactive CD8+T cells with high avidity for epitope V survive negative selection in SV11 mice but can be expanded by specific boosting approaches in the tumor bearing host.
Phenotype of a Herpes Simplex Virus Type 1 Mutant That Fails To Express Immediate-Early Regulatory Protein ICP0
Authors: Everett Roger D. , Boutell Chris , Orr Anne
Published: January 2004

Herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein ICP0 is required for efficient progression of infected cells into productive lytic infection, especially in low-multiplicity infections of limited-passage human fibroblasts. We have used single-cell-based assays that allow detailed analysis of the ICP0-null phenotype in low-multiplicity infections of restrictive cell types. The major conclusions are as follows: (i) there is a threshold input multiplicity above which the mutant virus replicates normally; (ii) individual cells infected below the threshold multiplicity have a high probability of establishing a nonproductive infection; (iii) such nonproductively infected cells have a high probability of expressing IE products at 6 h postinfection; (iv) even at 24 h postinfection, IE protein-positive nonproductively infected human fibroblast cells exceed the number of cells that lead to plaque formation by up to 2 orders of magnitude; (v) expression of individual IE proteins in a proportion of the nonproductively infected cells is incompletely coordinated; (vi) the nonproductive cells can also express early gene products at low frequencies and in a stochastic manner; and (vii) significant numbers of human fibroblast cells infected at low multiplicity by an ICP0-deficient virus are lost through cell death. We propose that in the absence of ICP0 expression, HSV-1 infected human fibroblasts can undergo a great variety of fates, including quiescence, stalled infection at a variety of different stages, cell death, and, for a minor population, initiation of formation of a plaque.
Identification of a Common HLA-DP4-Restricted T-Cell Epitope in the Conserved Region of the Respiratory Syncytial Virus G Protein
Authors: de Waal L. , Yüksel S. , Brandenburg A. H. , Langedijk J. P. M. , Sintnicolaas K. , Verjans G. M. G. M. , Osterhaus A. D. M. E. , de Swart R. L.
Published: January 2004

The cellular immune response to respiratory syncytial virus (RSV) is important in both protection and immunopathogenesis. In contrast to HLA class I, HLA class II-restricted RSV-specific T-cell epitopes have not been identified. Here, we describe the generation and characterization of two human RSV-specific CD4+-T-cell clones (TCCs) associated with type 0-like cytokine profiles. TCC 1 was specific for the matrix protein and restricted over HLA-DPB1*1601, while TCC 2 was specific for the attachment protein G and restricted over either HLA-DPB1*0401 or -0402. Interestingly, the latter epitope is conserved in both RSV type A and B viruses. Given the high allele frequencies of HLA-DPB1*0401 and -0402 worldwide, this epitope could be widely recognized and boosted by recurrent RSV infections. Indeed, peptide stimulation of peripheral blood mononuclear cells from healthy adults resulted in the detection of specific responses in 8 of 13 donors. Additional G-specific TCCs were generated from three of these cultures, which recognized the identical (n= 2) or almost identical (n= 1) HLA-DP4-restricted epitope as TCC 2. No significant differences were found between the capacities of cell lines obtained from infants with severe (n= 41) or mild (n= 46) RSV lower respiratory tract infections to function as antigen-presenting cells to the G-specific TCCs, suggesting that the severity of RSV disease is not linked to the allelic frequency of HLA-DP4. In conclusion, we have identified an RSV G-specific human T helper cell epitope restricted by the widely expressed HLA class II alleles DPB1*0401 and -0402. Its putative role in protection and/or immunopathogenesis remains to be determined.
Activation of the Early-Late Switch in Adenovirus Type 5 Major Late Transcription Unit Expression by L4 Gene Products
Authors: Farley Daniel C. , Brown Jason L. , Leppard Keith N.
Published: January 2004

The adenovirus major late transcription unit (MLTU) encodes multiple proteins from five regions, L1 to L5, through differential splicing and polyadenylation. MLTU expression is temporally regulated; only a single product from L1 (52/55K) is expressed prior to replication, but a subsequent switch, the mechanism of which has not been defined, leads to full expression that encompasses L1 IIIa and all L2 to L5 products. Transfection of a plasmid containing the complete MLTU gave a full array of proteins in proportions similar to those in a late infection, and in a time course, the temporal pattern of expression in a natural infection was reproduced. However, a plasmid truncated after the L3 poly(A) site exclusively expressed the L1 52/55K protein and was defective in the switch to full gene expression from L1 to L3. The L4 33K protein, supplied in
trans
, was sufficient to upregulate cytoplasmic mRNA for MLTU products characteristic of the late pattern of expression to levels comparable to those produced by the full-length MLTU. There was a corresponding increase in expression of the L1 IIIa, L2, and L3 proteins, except hexon. Hexon protein expression additionally required both the L4 100K protein in
trans
and sequences downstream of the L3 poly(A) site in
cis
. These results indicate that induction of L4 protein expression is a key event in the early-late switch in MLTU expression, which we propose is precipitated by small amounts of L4 expression in a feed-forward activation mechanism.
Nuclear Factor κB-Dependent Activation of the Antiapoptoticbfl-1Gene by the Epstein-Barr Virus Latent Membrane Protein 1 and Activated CD40 Receptor
Authors: D'Souza Brendan N. , Edelstein Leonard C. , Pegman Pamela M. , Smith Sinéad M. , Loughran Sinéad T. , Clarke Ann , Mehl Anja , Rowe Martin , Gélinas Céline , Walls Dermot
Published: January 2004

Suppression of the cellular apoptotic program by the oncogenic herpesvirus Epstein-Barr virus (EBV) is central to both the establishment of latent infection and the development of EBV-associated malignancies. We have previously shown that expression of the EBV latent membrane protein 1 (LMP1) in Burkitt's lymphoma cell lines leads to increased mRNA levels from the cellular antiapoptoticbfl-1gene (also known asA1). Furthermore, ectopic expression of Bfl-1 in an EBV-positive cell line exhibiting a latency type 1 infection protects against apoptosis induced by growth factor deprivation (B. N. D'Souza, M. Rowe, and D. Walls, J. Virol.74:6652-6658, 2000). We now report that LMP1 drivesbfl-1promoter activity through interactions with components of the tumor necrosis factor receptor (TNFR)/CD40 signaling pathway. We present evidence that this process is NF-κB dependent, involves the recruitment of TNFR-associated factor 2, and is mediated to a greater extent by the carboxyl-terminal activating region 2 (CTAR2) relative to the CTAR1 domain of LMP1. Activation of CD40 receptor also led to increasedbfl-1mRNA levels and an NF-κB-dependent increase inbfl-1promoter activity in Burkitt's lymphoma-derived cell lines. We have delineated a 95-bp region of the promoter that functions as an LMP1-dependent transcriptional enhancer in this cellular context. This sequence contains a novel NF-κB-like binding motif that is essential for transactivation ofbfl-1by LMP1, CD40, and the NF-κB subunit protein p65. These findings highlight the role of LMP1 as a mediator of EBV-host cell interactions and may indicate an important route by which it exerts its cellular growth transforming properties.
A Cytomegalovirus Inhibitor of Gamma Interferon Signaling Controls Immunoproteasome Induction
Authors: Khan Selina , Zimmermann Albert , Basler Michael , Groettrup Marcus , Hengel Hartmut
Published: January 2004

Both human and mouse cytomegaloviruses (HCMV and MCMV) avoid peptide presentation through the major histocompatibility complex (MHC) class I pathway to CD8+T cells. Within the MHC class I pathway, the vast majority of antigenic peptides are generated by the proteasome system, a multicatalytic protease complex consisting of constitutive subunits, three of which can be replaced by enzymatically active gamma interferon (IFN-γ)-inducible subunits, i.e., LMP2, LMP7, and MECL1, to form the so-called immunoproteasomes. Here, we show that steady-state levels of immunoproteasomes are readily formed in response to MCMV infection in the liver. In contrast, the incorporation of immunoproteasome subunits was prevented in MCMV-infected, as well as HCMV-infected, fibroblasts in vitro. Likewise, the expression of the IFN-γ-inducible proteasome regulator PA28αβ was also impaired in MCMV-infected cells. Both MCMV and HCMV did not alter the constitutive-subunit composition of proteasomes in infected cells. Quantitative assessment of LMP2, MECL1, and LMP7 transcripts revealed that the inhibition of immunoproteasome formation occurred at a pretranscriptional level. Remarkably, a targeted deletion of the MCMV geneM27, encoding an inhibitor of STAT2 that disrupts IFN-γ receptor signaling, largely restored transcription and protein expression of immunoproteasome subunits in infected cells. While CMV block peptide transport and MHC class I assembly by posttranslational strategies, immunoproteasome assembly, and thus the repertoire of proteasomal peptides, is controlled by pretranscriptional mechanisms. We hypothesize that the blockade of immunoproteasome formation has considerable consequences for shaping the CD8+-T-cell repertoire during the effector phase of the immune response.
Chimeric Human Immunodeficiency Virus Type 1 (HIV-1) Virions Containing HIV-2 or Simian Immunodeficiency Virus Nef Are Resistant to Cyclosporine Treatment
Authors: Khan Mahfuz , Jin Lingling , Huang Ming Bo , Miles Lesa , Bond Vincent C. , Powell Michael D.
Published: January 2004

The viral protein Nef and the cellular factor cyclophilin A are both required for full infectivity of human immunodeficiency virus type 1 (HIV-1) virions. In contrast, HIV-2 and simian immunodeficiency virus (SIV) do not incorporate cyclophilin A into virions or need it for full infectivity. Since Nef and cyclophilin A appear to act in similar ways on postentry events, we determined whether chimeric HIV-1 virions that contained either HIV-2 or SIV Nef would have a direct effect on cyclophilin A dependence. Our results show that chimeric HIV-1 virions containing either HIV-2 or SIV Nef are resistant to treatment by cyclosporine and enhance the infectivity of virions with mutations in the cyclophilin A binding loop of Gag. Amino acids at the C terminus of HIV-2 and SIV are necessary for inducing cyclosporine resistance. However, transferring these amino acids to the C terminus of HIV-1 Nef is insufficient to induce cyclosporine resistance in HIV-1. These results suggest that HIV-2 and SIV Nef are able to compensate for the need for cyclophilin A for full infectivity and that amino acids present at the C termini of these proteins are important for this function.
African Swine Fever Virus Multigene Family 360 and 530 Genes Affect Host Interferon Response
Authors: Afonso C. L. , Piccone M. E. , Zaffuto K. M. , Neilan J. , Kutish G. F. , Lu Z. , Balinsky C. A. , Gibb T. R. , Bean T. J. , Zsak L. , Rock D. L.
Published: January 2004

African swine fever virus (ASFV) multigene family 360 and 530 (MGF360/530) genes affect viral growth in macrophage cell cultures and virulence in pigs (L. Zsak, Z. Lu, T. G. Burrage, J. G. Neilan, G. F. Kutish, D. M. Moore, and D. L. Rock, J. Virol.75:3066-3076, 2001). The mechanism by which these novel genes affect virus-host interactions is unknown. To define MGF360/530 gene function, we compared macrophage transcriptional responses following infection with parental ASFV (Pr4) and an MGF360/530 deletion mutant (Pr4Δ35). A swine cDNA microarray containing 7,712 macrophage cDNA clones was used to compare the transcriptional profiles of swine macrophages infected with Pr4 and Pr4Δ35 at 3 and 6 h postinfection (hpi). While at 3 hpi most (7,564) of the genes had similar expression levels in cells infected with either virus, 38 genes had significantly increased (>2.0-fold,P< 0.05) mRNA levels in Pr4Δ35-infected macrophages. Similar up-regulation of these genes was observed at 6 hpi. Viral infection was required for this induced transcriptional response. Most Pr4Δ35 up-regulated genes were part of a type I interferon (IFN) response or were genes that are normally induced by double-stranded RNA and/or viral infection. These included monocyte chemoattractant protein, transmembrane protein 3, tetratricopeptide repeat protein 1, a ubiquitin-like 17-kDa protein, ubiquitin-specific protease ISG43, an RNA helicase DEAD box protein, GTP-binding MX protein, the cytokine IP-10, and the PKR activator PACT. Differential expression of IFN early-response genes in Pr4Δ35 relative to Pr4 was confirmed by Northern blot analysis and real-time PCR. Analysis of IFN-α mRNA and secreted IFN-α levels at 3, 8, and 24 hpi revealed undetectable IFN-α in mock- and Pr4-infected macrophages but significant IFN-α levels at 24 hpi in Pr4Δ35-infected macrophages. The absence of IFN-α in Pr4-infected macrophages suggests that MGF360/530 genes either directly or indirectly suppress a type I IFN response. An inability to suppress host type I IFN responses may account for the growth defect of Pr4Δ35 in macrophages and its attenuation in swine.
Glycine Decarboxylase Mediates a Postbinding Step in Duck Hepatitis B Virus Infection
Authors: Li Jisu , Tong Shuping , Lee Hong Bock , Perdigoto Ana Luisa , Spangenberg Hans Christian , Wands Jack R.
Published: January 2004

Envelope protein precursors of many viruses are processed by a basic endopeptidase to generate two molecules, one for receptor binding and the other for membrane fusion. Such a cleavage event has not been demonstrated for the hepatitis B virus family. Two binding partners for duck hepatitis B virus (DHBV) pre-S envelope protein have been identified. Duck carboxypeptidase D (DCPD) interacts with the full-length pre-S protein and is the DHBV docking receptor, while duck glycine decarboxylase (DGD) has the potential to bind several deletion constructs of the pre-S protein in vitro. Interestingly, DGD but not DCPD expression was diminished following prolonged culture of primary duck hepatocytes (PDH), which impaired productive DHBV infection. Introduction of exogenous DGD promoted formation of protein-free viral genome, suggesting restoration of several early events in viral life cycle. Conversely, blocking DGD expression in fresh PDH by antisense RNA abolished DHBV infection. Moreover, addition of DGD antibodies soon after virus binding reduced endogenous DGD protein levels and impaired production of covalently closed circular DNA, the template for DHBV gene expression and genome replication. Our findings implicate this second pre-S binding protein as a critical cellular factor for productive DHBV infection. We hypothesize that DCPD, a molecule cycling between the cell surface and the
trans
-Golgi network, targets DHBV particles to the secretary pathway for proteolytic cleavage of viral envelope protein. DGD represents the functional equivalent of other virus receptors in its interaction with processed viral particles.
Reovirus Nonstructural Protein μNS Recruits Viral Core Surface Proteins and Entering Core Particles to Factory-Like Inclusions
Authors: Broering Teresa J. , Kim Jonghwa , Miller Cathy L. , Piggott Caroline D. S. , Dinoso Jason B. , Nibert Max L. , Parker John S. L.
Published: January 2004

Mammalian reoviruses are thought to assemble and replicate within cytoplasmic, nonmembranous structures called viral factories. The viral nonstructural protein μNS forms factory-like globular inclusions when expressed in the absence of other viral proteins and binds to the surfaces of the viral core particles in vitro. Given these previous observations, we hypothesized that one or more of the core surface proteins may be recruited to viral factories through specific associations with μNS. We found that all three of these proteins—λ1, λ2, and σ2—localized to factories in infected cells but were diffusely distributed through the cytoplasm and nucleus when each was separately expressed in the absence of other viral proteins. When separately coexpressed with μNS, on the other hand, each core surface protein colocalized with μNS in globular inclusions, supporting the initial hypothesis. We also found that λ1, λ2, and σ2 each localized to filamentous inclusions formed upon the coexpression of μNS and μ2, a structurally minor core protein that associates with microtubules. The first 40 residues of μNS, which are required for association with μ2 and the RNA-binding nonstructural protein σNS, were not required for association with any of the three core surface proteins. When coexpressed with μ2 in the absence of μNS, each of the core surface proteins was diffusely distributed and displayed only sporadic, weak associations with μ2 on filaments. Many of the core particles that entered the cytoplasm of cycloheximide-treated cells following entry and partial uncoating were recruited to inclusions of μNS that had been preformed in those cells, providing evidence that μNS can bind to the surfaces of cores in vivo. These findings expand a model for how viral and cellular components are recruited to the viral factories in infected cells and provide further evidence for the central but distinct roles of viral proteins μNS and μ2 in this process.
Lytic Induction Therapy for Epstein-Barr Virus-Positive B-Cell Lymphomas
Authors: Feng Wen-hai , Hong Gregory , Delecluse Henri-Jacques , Kenney Shannon C.
Published: January 2004

A novel therapy for Epstein-Barr virus (EBV)-positive tumors involves the intentional induction of the lytic form of EBV infection combined with ganciclovir (GCV) treatment. Virally encoded kinases (thymidine kinase and BGLF4) which are expressed only during the lytic form of infection convert GCV (a nucleoside analogue) into its active, cytotoxic form. However, tightly latent EBV infection in B cells has made it difficult to identify drugs that can be used clinically to induce lytic viral infection in B-cell lymphomas. Here we demonstrate that gemcitabine and doxorubicin (but not 5-azacytidine,
cis
-platinum, or 5-fluorouracil) induce lytic EBV infection in EBV-transformed B cells in vitro and in vivo. Gemcitabine and doxorubicin both activated transcription from the promoters of the two viral immediate-early genes, BZLF1 and BRLF1, in EBV-negative B cells. This effect required the EGR-1 motif in the BRLF1 promoter and the CRE (ZII) and MEF-2D (ZI) binding sites in the BZLF1 promoter. GCV enhanced cell killing by gemcitabine or doxorubicin in lymphoblastoid cells transformed with wild-type EBV, but not in lymphoblastoid cells transformed by a mutant virus (with a deletion in the BZLF1 immediate-early gene) that is unable to enter the lytic form of infection. Most importantly, the combination of gemcitabine or doxorubicin and GCV was significantly more effective for the inhibition of EBV-driven lymphoproliferative disease in SCID mice than chemotherapy alone. In contrast, the combination of zidovudine and gemcitabine was no more effective than gemcitabine alone. These results suggest that the addition of GCV to either gemcitabine- or doxorubicin-containing chemotherapy regimens may enhance the therapeutic efficacy of these drugs for EBV-driven lymphoproliferative disease in patients.
Formation of Nuclear Foci of the Herpes Simplex Virus Type 1 Regulatory Protein ICP4 at Early Times of Infection: Localization, Dynamics, Recruitment of ICP27, and Evidence for the De Novo Induction of ND10-Like Complexes
Authors: Everett Roger D. , Sourvinos George , Leiper Claire , Clements J. Barklie , Orr Anne
Published: January 2004

Herpes simplex virus type 1 (HSV-1) has an intricate association with cellular nuclear structures known as ND10 or promyelocytic leukemia protein (PML) nuclear bodies. Parental viral genomes initially become juxtaposed to ND10, and then viral replication compartments develop from the ND10-associated genomes. Viral immediate-early (IE) regulatory protein ICP0 colocalizes with ND10 and then induces the degradation of critical ND10 component protein PML and therefore the release and dispersal of other ND10 proteins. The IE transcriptional regulatory protein ICP4 also forms foci at early times of infection, many of which are juxtaposed to ND10 and later develop into replication compartments, indicating that at least some of the initial ICP4 foci contain parental viral genomes. Here we report that the ICP4 foci also contain ICP27 and that their formation occurs extremely rapidly at locations just inside the nuclear envelope. By examining developing plaques or thinly seeded cells infected at high multiplicity, we found evidence to suggest that at least some of the ND10-viral nucleoprotein complex association could be attributed to de novo formation of ND10-like structures in response to incoming viral genomes. The ICP4 complexes associated efficiently with ND10 in cells infected with an ICP0-null mutant virus at high but not at low multiplicity, and the degree of association was reduced by the proteasome inhibitor MG132. Therefore, the interaction between viral nucleoprotein complexes and ND10 is in part due to a dynamic response by the cell. This response is modulated by functional ICP0, and cells that are productively or nonproductively infected in the absence of functional ICP0 can be distinguished by the relative locations of ICP4 foci and ND10 proteins.
The K1 Protein of Kaposi's Sarcoma-Associated Herpesvirus Activates the Akt Signaling Pathway
Authors: Tomlinson Christine C. , Damania Blossom
Published: January 2004

Kaposi's sarcoma-associated herpesvirus (KSHV) has been implicated in Kaposi's sarcoma, as well as in primary effusion lymphoma and multicentric Castleman's disease. The K1 protein of KSHV has been shown to induce cellular transformation and focus formation and to deregulate B-lymphocyte signaling pathways by functionally mimicking the activated B-cell receptor complex. Here we show that expression of K1 in B lymphocytes targets the phosphatidylinositol-3 kinase pathway, leading to the activation of the Akt kinase and the inhibition of the phosphatase PTEN. We also demonstrate that activation of Akt by the K1 protein leads to the phosphorylation and inhibition of members of the forkhead (FKHR) transcription factor family, which are key regulators of cell cycle progression and apoptosis. We demonstrate that K1 can inhibit apoptosis induced by the FKHR proteins and by stimulation of the Fas receptor. Our observations suggest that the K1 viral protein promotes cell survival pathways and may contribute to KSHV pathogenesis by preventing virally infected cells from undergoing apoptosis prematurely.
Bending of Adenovirus Origin DNA by Nuclear Factor I as Shown by Scanning Force Microscopy Is Required for Optimal DNA Replication
Authors: Mysiak Monika E. , Bleijenberg Marjoleine H. , Wyman Claire , Holthuizen P. Elly , van der Vliet Peter C.
Published: January 2004

Nuclear factor I (NFI) is a transcription factor that binds to the adenovirus type 5 (Ad5) origin of replication and recruits the adenovirus DNA polymerase, thereby stimulating initiation of DNA replication in vitro. Using scanning force microscopy, we demonstrate that NFI induces a 60° bend upon binding to the origin. The A/T-rich region preceding the core recognition sequence of NFI influences the DNA bend angle, since substitution of A/T base pairs by G/C base pairs severely decreases bending. Mutations in the A/T-rich region do not affect binding of NFI to DNA. However, mutations that reduce the protein-induced bend lead to a loss of NFI-stimulated replication, indicating that DNA bending is functionally important. In contrast, basal initiation or DNA binding of the polymerase is not impaired by these origin mutations. We conclude that binding of NFI to the Ad5 origin causes structural changes in DNA that are essential for the stimulatory function of NFI in replication. We propose that NFI-induced origin bending facilitates the assembly of a functional initiation complex.
Reversible Inhibition of Spreading of In Vitro Infection and Imbalance of Viral Protein Accumulation at Low pH in Viral Hemorrhagic Septicemia Rhabdovirus, a Salmonid Rhabdovirus
Authors: Mas V. , Rocha A. , Perez L. , Coll J. M. , Estepa A.
Published: January 2004

The inhibition of viral hemorrhagic septicemia rhabdovirus (VHSV) in vitro infection by pHs of <7 (low pH) has been previously reported. Nevertheless, the details of the mechanism underlying this effect remain obscure. We present evidence showing that low-pH inhibition occurs during a viral postadsorption step. Thus, while VHSV bound, replicated within single cells, and presented its G protein on the membranes of infected cells at both low and physiological pHs, both cell-to-cell spreading of infection (as estimated by the appearance of foci of infected cells) and fusion (as estimated by a syncytium assay) were inhibited by this low pH. The decreased VHSV titers and the inhibition of both cell-to-cell spreading of infection and fusion could be reversed by adjusting the pH to 7.5 at any time during infection. This effect should be taken into account to avoid false negatives in the diagnosis of VHSV by cell culture. On the other hand, the cell-to-cell spreading of infection at pH 7.5 could be stopped at any time by reducing the pH to 6.5. Since at low pH there were changes in the protein G conformation and smaller and imbalanced amounts of N with respect to M1, M2, and G viral proteins, alterations of the assembly and/or budding of VHSV are most probably involved in the absence of newly released infective virions.
Identification of Antigenic Regions of Duck Hepatitis B Virus Core Protein with Antibodies Elicited by DNA Immunization and Chronic Infection
Authors: Thermet A. , Robaczewska M. , Rollier C. , Hantz O. , Trepo C. , Deleage G. , Cova L.
Published: January 2004

The induction of humoral response in ducks by DNA-based immunization against duck hepatitis B virus (DHBV) core protein (DHBc) was investigated. In addition, the amino acid specificity of the induced response was compared by using peptide scanning to that elicited either by protein immunization or during chronic DHBV infection. Immunization of ducks with a plasmid expressing DHBc protein led to the induction of a long-lasting antibody response able to specifically recognize viral protein in chronically infected duck livers. Peptide scanning analysis of anti-DHBc response induced during chronic DHBV infection allowed us to identify six major antigenic regions (AR1 to AR6). The reactivity spectrum of duck sera elicited by protein immunization appeared narrower and was restricted to only four of these antigenic regions in spite of higher anti-DHBc antibody titers. Interestingly, anti-DHBc antibodies induced by DNA-based immunization recognized five of six antigenic regions, and the epitope pattern was broader and more closely related to that observed in chronic viral infections. To gain more insight into the location of antigenic regions, we built a three-dimensional (3-D) model of DHBc protein based on human and duck core sequence alignment data and the HBc 3-D crystal structure. The results suggest that two identified antigenic regions (AR2, amino acids [aa]64T-P84, and AR5, aa183A-R210) are located at positions on the protein surface equivalent to those of the two HBc major epitopes. Moreover, we identified another antigenic region (AR3, aa99I-I112) that was recognized by all sera from chronically infected, DNA- or protein-immunized ducks within the large 45-aa insertion in DHBc protein, suggesting that this region, which lacks HBc, is externally exposed.
Comparative Genomic Analysis of Hyperthermophilic Archaeal
Fuselloviridae
Viruses
Authors: Wiedenheft Blake , Stedman Kenneth , Roberto Francisco , Willits Deborah , Gleske Anne-Kathrin , Zoeller Luisa , Snyder Jamie , Douglas Trevor , Young Mark
Published: January 2004

The complete genome sequences of two
Sulfolobus
spindle-shaped viruses (SSVs) from acidic hot springs in Kamchatka (Russia) and Yellowstone National Park (United States) have been determined. These nonlytic temperate viruses were isolated from hyperthermophilic
Sulfolobus
hosts, and both viruses share the spindle-shaped morphology characteristic of the
Fuselloviridae
family. These two genomes, in combination with the previously determined SSV1 genome from Japan and the SSV2 genome from Iceland, have allowed us to carry out a phylogenetic comparison of these geographically distributed hyperthermal viruses. Each virus contains a circular double-stranded DNA genome of ∼15 kbp with approximately 34 open reading frames (ORFs). These
Fusellovirus
ORFs show little or no similarity to genes in the public databases. In contrast, 18 ORFs are common to all four isolates and may represent the minimal gene set defining this viral group. In general, ORFs on one half of the genome are colinear and highly conserved, while ORFs on the other half are not. One shared ORF among all four genomes is an integrase of the tyrosine recombinase family. All four viral genomes integrate into their host tRNA genes. The specific tRNA gene used for integration varies, and one genome integrates into multiple loci. Several unique ORFs are found in the genome of each isolate.
Comparative Study of Adaptive Molecular Evolution in Different Human Immunodeficiency Virus Groups and Subtypes
Authors: Choisy Marc , Woelk Christopher H. , Guégan Jean-François , Robertson David L.
Published: January 2004

Molecular adaptation, as characterized by the detection of positive selection, was quantified in a number of genes from different human immunodeficiency virus type 1 (HIV-1) group M subtypes, group O, and an HIV-2 subtype using the codon-based maximum-likelihood method of Yang and coworkers (Z. H. Yang, R. Nielsen, N. Goldman, and A. M. K. Pedersen, Genetics 155:431-449, 2000). Theenvgene was investigated further since it exhibited the strongest signal for positive selection compared to those of the other two major HIV genes (gagandpol). In order to investigate the pattern of adaptive evolution acrossenv, the location and strength of positive selection in different HIV-1 sequence alignments was compared. The number of sites having a significant probability of being positively selected varied among these different alignment data sets, ranging from 25 in HIV-1 group M subtype A to 40 in HIV-1 group O. Strikingly, there was a significant tendency for positively selected sites to be located at the same position in different HIV-1 alignments, ranging from 10 to 16 shared sites for the group M intersubtype comparisons and from 6 to 8 for the group O to M comparisons, suggesting that all HIV-1 variants are subject to similar selective forces. As the host immune response is believed to be the dominant driving force of adaptive evolution in HIV, this result would suggest that the same sites are contributing to viral persistence in diverse HIV infections. Thus, the positions of the positively selected sites were investigated in reference to the inferred locations of different epitope types (antibody, T helper, and cytotoxic T lymphocytes) and the positions of N and O glycosylation sites. We found a significant tendency for positively selected sites to fall outside T-helper epitopes and for positively selected sites to be strongly associated with N glycosylation sites.