bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleic Acid-Dependent Cross-Linking of the Nucleocapsid Protein of Sindbis Virus
Authors: Tellinghuisen Timothy L. , Kuhn Richard J.
Published: July 2002

The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway.
Brome Mosaic Virus Polymerase-Like Protein 2a Is Directed to the Endoplasmic Reticulum by Helicase-Like Viral Protein 1a
Authors: Chen Jianbo , Ahlquist Paul
Published: July 2002

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes RNA replication proteins 1a and 2a. 1a contains a C-terminal helicase-like domain and an N-terminal domain implicated in viral RNA capping, and 2a contains a central polymerase-like domain. 1a and 2a colocalize in an endoplasmic reticulum (ER)-associated replication complex that is the site of BMV-specific RNA-dependent RNA synthesis in plant and yeast cells. 1a also localizes to the ER in the absence of 2a or viral RNA replication templates. To investigate the determinants of 2a localization, we fused 2a to the green fluorescent protein (GFP), creating a functional GFP-2a fusion that supported BMV RNA replication and subgenomic mRNA transcription. In the absence of 1a, the GFP-2a fusion was found to be diffused throughout the cytoplasm and in punctate spots not associated with any cytoplasmic organelle so far tested. Formation of these spots was dependent on the C-terminal half of 2a and may represent aggregation of a fraction of 2a. When coexpressed with 1a, GFP-2a colocalized with 1a and ER-resident protein Kar2p in a partial or complete ring around the nucleus. Consistent with these results, cell fractionation showed that both the GFP-2a fusion and wild-type (wt) 2a remained soluble when expressed alone, while in cells coexpressing 1a, most of the GFP-2a fusion or wt 2a cofractionated with 1a in the rapidly sedimenting membrane fraction. Deletion analysis showed that the N-terminal 120-amino-acid segment of 2a, containing one of two 2a regions previously shown to interact with 1a, was necessary and sufficient for 1a-directed localization of GFP-2a derivatives to the ER. These results suggest that 1a, which also interacts independently with the ER and viral RNA, is a key organizer of RNA replication complex assembly.
Persistence of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T-Lymphocyte Clones in a Subject with Rapid Disease Progression
Authors: Islam Sabina A. , Hay Christine M. , Hartman Kelly E. , He Suqin , Shea Amy K. , Trocha Alicja K. , Dynan Mark J. , Reshamwala Neha , Buchbinder Susan P. , Basgoz Nesli O. , Kalams Spyros A.
Published: July 2002

We longitudinally measured T-cell receptor transcript frequencies of human immunodeficiency virus type 1 (HIV-1) specific cytotoxic T lymphocytes (CTL) in an individual with rapidly progressive disease and high levels of viremia. CTL clones elicited during acute HIV-1 infection were present at the time of death, despite absent functional CTL responses, arguing against clonal deletion as a mechanism for the decline of CTL responses observed during HIV-1 infection.
The Adenovirus Type 5 E1B-55K Oncoprotein Actively Shuttles in Virus-Infected Cells, Whereas Transport of E4orf6 Is Mediated by a CRM1-Independent Mechanism
Authors: Dosch Tanja , Horn Florian , Schneider Grit , Krätzer Friedrich , Dobner Thomas , Hauber Joachim , Stauber Roland H.
Published: July 2002

The E1B-55K and E4orf6 proteins of adenovirus type 5 are involved in viral mRNA export. Here we demonstrate that adenovirus infection does not inhibit the function of the E1B-55K nuclear export signal and that E1B-55K also shuttles in infected cells. Even during virus infection, E1B-55K was exported by the leptomycin B-sensitive CRM1 pathway, whereas E4orf6 transport appeared to be mediated by an alternative mechanism. Our results strengthen the potential role of E1B-55K as the “driving force” for adenoviral late mRNA export.
Development of an Avian Leukosis-Sarcoma Virus Subgroup A Pseudotyped Lentiviral Vector
Authors: Lewis Brian C. , Chinnasamy Nachimuthu , Morgan Richard A. , Varmus Harold E.
Published: July 2002

We are using avian leukosis-sarcoma virus (ALSV) vectors to generate mouse tumor models in transgenic mice expressing TVA, the receptor for subgroup A ALSV. Like other classical retroviruses, ALSV requires cell division to establish a provirus after infection of host cells. In contrast, lentiviral vectors are capable of integrating their viral DNA into the genomes of nondividing cells. With the intention of initiating tumorigenesis in resting, TVA-positive cells, we have developed a system for the preparation of a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector, pseudotyped with the envelope protein of ALSV subgroup A (EnvA). The HIV(ALSV-A) vector retains the requirement for TVA on the surface of target cells and can be produced at titers of 5 × 10
3
infectious units (IU)/ml. By inserting the central polypurine tract (cPPT) from the HIV-1
pol
gene and removing the cytoplasmic tail of EnvA, the pseudotype can be produced at titers approaching 10
5
IU/ml and can be concentrated by ultracentrifugation to titers of 10
7
IU/ml. HIV(ALSV-A) also infects embryonic fibroblasts derived from transgenic mice in which TVA expression is driven by the β-actin promoter. In addition, this lentivirus pseudotype efficiently infects these fibroblasts after cell cycle arrest, when they are resistant to infection by ALSV vectors. This system may be useful for introducing genes into somatic cells in adult TVA transgenic animals and allows evaluation of the effects of altered gene expression in differentiated cell types in vivo.
The Bovine Herpesvirus 1 Immediate-Early Protein (bICP0) Associates with Histone Deacetylase 1 To Activate Transcription
Authors: Zhang Yange , Jones Clinton
Published: July 2002

Infected-cell protein 0 encoded by bovine herpesvirus 1 (BHV-1) (bICP0) is necessary for efficient productive infection, in large part, because it activates all 3 classes of BHV-1 genes (U. V. Wirth, C. Fraefel, B. Vogt, C. Vlcek, V. Paces, and M. Schwyzer, J. Virol. 66:2763–2772, 1992). Although bICP0 is believed to be a functional homologue of herpes simplex virus type 1-encoded ICP0, the only well-conserved domain between the proteins is a zinc ring finger located near the amino terminus of both proteins. Our previous studies demonstrated that bICP0 is toxic to transfected cells but does not appear to directly induce apoptosis (Inman, M., Y. Zhang, V. Geiser, and C. Jones, J. Gen. Virol. 82:483–492, 2001). C-terminal sequences in the last 320 amino acids of bICP0 mediate subcellular localization. Mutagenesis of the zinc ring finger within bICP0 revealed that this domain was important for transcriptional activation. In this study, we demonstrate that bICP0 interacts with histone deacetylase 1 (HDAC1), which results in activation of a simple promoter containing four consensus Myc-Max binding sites. The interaction between bICP0 and HDAC1 correlated with inhibition of Mad-dependent transcriptional repression. In resting CV-1 cells, bICP0 relieved HDAC1-mediated transcriptional repression. The zinc ring finger was required for relieving HDAC1-induced repression but not for interacting with HDAC1. In fetal bovine lung cells but not in a human epithelial cell line, bICP0 expression correlated with reduced steady-state levels of HDAC1 in crude cytoplasmic extracts. We hypothesize that the ability of bICP0 to overcome HDAC1-induced repression plays a role in promoting productive infection in highly differentiated cell types.
Functional Murine Leukemia Virus Vectors Pseudotyped with the Visna Virus Envelope Show Expanded Visna Virus Cell Tropism
Authors: Bruett Linda , Clements Janice E.
Published: July 2002

Pseudotype virus vectors serve as a powerful tool for the study of virus receptor usage and entry. We describe the development of murine leukemia virus (MuLV) particles pseudotyped with the visna virus envelope glycoprotein and encoding a green fluorescent protein reporter as a tool to study the expression of the visna virus receptor. Functional MuLV/visna virus pseudotypes were obtained when the cytoplasmic tail of the visna virus envelope TM protein was truncated to 3, 7, or 11 amino acids in length. MuLV/visna virus particles were used to transduce a panel of cell types from various organisms, including sheep, goat, human, hamster, mouse, monkey, and quail. The majority of the cells examined were susceptible to MuLV/visna pseudotype viruses, supporting the notion that the visna virus cellular receptor is a widely expressed protein found in many species. Of 16 different cell types tested, only mouse embryo fibroblast NIH 3T3 cells, hamster ovary CHO cells, and the human promonocyte cell line U937 cells were not susceptible to transduction by the pseudotyped virus. The production of functional MuLV/visna virus pseudotypes has provided a sensitive, biologically relevant system to study visna virus cell entry and envelope-receptor interactions.
Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein
Authors: Rothenberg S. Michael , Olsen Mari N. , Laurent Louise Chang , Crowley Rachel Adams , Brown Patrick O.
Published: July 2002

The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the
env
gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.
Cap-Independent Translation Conferred by the 5′ Leader of Tobacco Etch Virus Is Eukaryotic Initiation Factor 4G Dependent
Authors: Gallie Daniel R.
Published: July 2002

The 5′ leader of tobacco etch virus (TEV) genomic RNA directs efficient translation from the naturally uncapped viral mRNA. Two distinct regions within the TEV 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mRNA, indicating that the TEV leader contains an internal ribosome entry site (IRES). In this study, the requirements for TEV IRES activity were investigated. The TEV IRES enhanced translation of monocistronic or dicistronic mRNAs in vitro under competitive conditions, i.e., at high RNA concentration or in lysate partially depleted of eukaryotic initiation factor 4F (eIF4F) and eIFiso4F, the two cap binding complexes in plants. The translational advantage conferred by the TEV IRES under these conditions was lost when the lysate reduced in eIF4F and eIFiso4F was supplemented with eIF4F (or, to a lesser extent, eIFiso4F) but not when supplemented with eIF4E, eIFiso4E, eIF4A, or eIF4B. eIF4G, the large subunit of eIF4F, was responsible for the competitive advantage conferred by the TEV IRES. TEV IRES activity was enhanced moderately by the poly(A)-binding protein. These observations suggest that the TEV IRES directs cap-independent translation through a mechanism that involves eIF4G specifically.
Neutralization Synergy of Human Immunodeficiency Virus Type 1 Primary Isolates by Cocktails of Broadly Neutralizing Antibodies
Authors: Zwick Michael B. , Wang Meng , Poignard Pascal , Stiegler Gabriela , Katinger Hermann , Burton Dennis R. , Parren Paul W. H. I.
Published: July 2002

Several reports have described the existence of synergy between neutralizing monoclonal antibodies (MAbs) against human immunodeficiency virus type 1 (HIV-1). Synergy between human MAbs b12, 2G12, 2F5, and 4E10 in neutralization of primary isolates is of particular interest. Neutralization synergy of these MAbs, however, has not been studied extensively, and the mechanism of synergy remains unclear. We investigated neutralization synergy among this human antibody set by using the classical approach of titrating antibodies mixed at a fixed ratio as well as by an alternative, variable ratio approach in which the neutralization curve of one MAb is assessed in the presence and absence of a fixed, weakly neutralizing concentration of a second antibody. The advantage of this second approach is that it does not require mathematical analysis to establish synergy. No neutralization enhancement of any of the MAb combinations tested was detected for the T-cell-line-adapted molecular HIV-1 clone HxB2 using both assay formats. Studies of primary isolates (89.6, SF162, and JR-CSF) showed neutralization synergy which was relatively weak, with a maximum of two- to fourfold enhancement between antibody pairs, thereby increasing neutralization titers about 10-fold in triple and quadruple antibody combinations. Analysis of b12 and 2G12 binding to oligomeric envelope glycoprotein by using flow cytometry failed to demonstrate cooperativity in binding between these two antibodies. The mechanism by which these antibodies synergize is, therefore, not yet understood. The results lend some support to the notion that an HIV-1 vaccine that elicits moderate neutralizing antibodies to multiple epitopes may be more effective than hereto supposed, although considerable caution in extrapolating to a vaccine situation is required.
Polyvalent Envelope Glycoprotein Vaccine Elicits a Broader Neutralizing Antibody Response but Is Unable To Provide Sterilizing Protection against Heterologous Simian/Human Immunodeficiency Virus Infection in Pigtailed Macaques
Authors: Cho Michael W. , Kim Young B. , Lee Myung K. , Gupta Kailash C. , Ross Will , Plishka Ron , Buckler-White Alicia , Igarashi Tatsuhiko , Theodore Ted , Byrum Russ , Kemp Chris , Montefiori David C. , Martin Malcolm A.
Published: July 2002

The great difficulty in eliciting broadly cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) isolates has been attributed to several intrinsic properties of their viral envelope glycoprotein, including its complex quaternary structure, extensive glycosylation, and marked genetic variability. Most previously evaluated vaccine candidates have utilized envelope glycoprotein from a single virus isolate. Here we compare the breadth of NAb and protective immune response following vaccination of pigtailed macaques with envelope protein(s) derived from either single or multiple viral isolates. Animals were challenged with Simian/human immunodeficiency virus strain DH12 (SHIV
DH12
) following priming with recombinant vaccinia virus(es) expressing gp160(s) and boosting with gp120 protein(s) from (i) LAI, RF, 89.6, AD8, and Bal (Polyvalent); (ii) LAI, RF, 89.6, AD8, Bal, and DH12 (Polyvalent-DH12); (iii) 89.6 (Monovalent-89.6); and (iv) DH12 (Monovalent-DH12). Animals in the two polyvalent vaccine groups developed NAbs against more HIV-1 isolates than those in the two monovalent vaccine groups (
P
= 0.0054). However, the increased breadth of response was directed almost entirely against the vaccine strains. Resistance to SHIV
DH12
strongly correlated with the level of NAbs directed against the virus on the day of challenge (
P
= 0.0008). Accordingly, the animals in the Monovalent-DH12 and Polyvalent-DH12 vaccine groups were more resistant to the SHIV
DH12
challenge than the macaques immunized with preparations lacking a DH12 component (viz. Polyvalent and Monovalent-89.6) (
P
= 0.039). Despite the absence of any detectable NAb, animals in the Polyvalent vaccine group, but not those immunized with Monovalent-89.6, exhibited markedly lower levels of plasma virus than those in the control group, suggesting a superior cell-mediated immune response induced by the polyvalent vaccine.
Epstein-Barr Virus
Bam
HI-A Rightward Transcript-Encoded RPMS Protein Interacts with the CBF1-Associated Corepressor CIR To Negatively Regulate the Activity of EBNA2 and NotchIC
Authors: Zhang Jinxia , Chen Honglin , Weinmaster Gerry , Hayward S. Diane
Published: July 2002

The Epstein-Barr virus (EBV)
Bam
HI-A rightward transcripts (BARTs) are expressed in all EBV-associated tumors as well as in latently infected B cells in vivo and cultured B-cell lines. One of the BART family transcripts contains an open reading frame, RPMS1, that encodes a nuclear protein termed RPMS. Reverse transcription-PCR analysis revealed that BART transcripts with the splicing pattern that generates the RPMS1 open reading frame are commonly expressed in EBV-positive lymphoblastoid cell lines and are also detected in Hodgkin's disease tissues. Experiments undertaken to determine the function of RPMS revealed that RPMS interacts with both CBF1 and components of the CBF1-associated corepressor complex. RPMS interaction with CBF1 was demonstrated in a glutathione
S
-transferase (GST) affinity assay and by the ability of RPMS to alter the intracellular localization of a mutant CBF1. A Gal4-RPMS fusion protein mediated transcriptional repression, suggesting an additional interaction between RPMS and corepressor proteins. GST affinity assays revealed interaction between RPMS and the corepressor Sin3A and CIR. The RPMS-CIR interaction was further substantiated in mammalian two-hybrid, coimmunoprecipitation, and colocalization experiments. RPMS has been shown to interfere with NotchIC and EBNA2 activation of CBF1-containing promoters in reporter assays. Consistent with this function, immunofluorescence assays performed on cotransfected cells showed that there was colocalization of RPMS with NotchIC and with EBNA2 in intranuclear punctate speckles. The effect of RPMS on NotchIC function was further examined in a muscle cell differentiation assay where RPMS was found to partially reverse NotchIC-mediated inhibition of differentiation. The mechanism of RPMS action was examined in cotransfection and mammalian two-hybrid assays. The results revealed that RPMS blocked relief of CBF1-mediated repression and interfered with SKIP-CIR interactions. We conclude that RPMS acts as a negative regulator of EBNA2 and Notch activity through its interactions with the CBF1-associated corepressor complex.
Establishment of Latent Epstein-Barr Virus Infection and Stable Episomal Maintenance in Murine B-Cell Lines
Authors: Haan Keith M. , Aiyar Ashok , Longnecker Richard
Published: July 2002

Epstein-Barr virus (EBV) is a strict human pathogen for which no small animal models exist. Plasmids that contain the EBV plasmid origin of replication,oriP, and express EBV nuclear antigen 1 (EBNA1) are stably maintained extrachromosomally in human cells, whereas these plasmids replicate poorly in rodent cells. However, the ability oforiPand EBNA1 to maintain the entire EBV episome in proliferating rodent cells has not been determined. Expression of the two human B-cell receptors for EBV on the surfaces of murine B cells allows efficient viral entry that leads to the establishment of latent EBV infection and long-term persistence of the viral genome. Latent gene expression in these cells resembles the latency II profile in that EBNA1 and LMP1 can be detected whereas EBNA2 and the EBNA3s are not expressed.
Hepatitis B Virus X Protein Acts as a Tumor Promoter in Development of Diethylnitrosamine-Induced Preneoplastic Lesions
Authors: Madden Charles R. , Finegold Milton J. , Slagle Betty L.
Published: July 2002

Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma. Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens. Although the mechanism for this cofactor role remains unknown, the ability of HBx to inhibit DNA repair and to influence cell cycle progression suggests two possible pathways. To investigate these possibilities in vivo, we treated double-transgenic mice that both express HBx (ATX mice) and possess a bacteriophage lambda transgene with the hepatocarcinogen diethylnitrosamine (DEN). Histological examination of liver tissue confirmed that DEN-treated ATX mice developed approximately twice as many focal lesions of basophilic hepatocytes as treated wild-type littermates. Treatment of mice with DEN resulted in a six- to eightfold increase in the mutation frequency (MF), as measured by a functional analysis of the lambda transgene. HBx expression was confirmed by immunoprecipitation and Western blotting and was associated with a modest 23% increase in the MF. Importantly, the extent of hepatocellular proliferation in 14-day-old mice, as measured by the detection of proliferating cell nuclear antigen and by the incorporation of 5-bromo-2′-deoxyuridine, was determined to be approximately twofold higher in ATX livers than in wild-type livers. These results are consistent with a model in which HBx expression contributes to the development of DEN-mediated carcinogenesis by promoting the proliferation of altered hepatocytes rather than by directly interfering with the repair of DNA lesions.
Pseudotyping
Autographa californica
Multicapsid Nucleopolyhedrovirus (Ac
M
NPV): F Proteins from Group II NPVs Are Functionally Analogous to Ac
M
NPV GP64
Authors: Lung Oliver , Westenberg Marcel , Vlak Just M. , Zuidema Douwe , Blissard Gary W.
Published: July 2002

GP64, the major envelope glycoprotein of budded virions of the baculovirus
Autographa californica
multicapsid nucleopolyhedrovirus (Ac
M
NPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral propagation in cell culture and in animals. Recent genome sequences from a number of baculoviruses show that only a subset of closely related baculoviruses have
gp64
genes, while other baculoviruses have a recently discovered unrelated envelope protein named F. F proteins from
Lymantria dispar M
NPV (Ld
M
NPV) and
Spodoptera exigua M
NPV (Se
M
NPV) mediate membrane fusion and are therefore thought to serve roles similar to that of GP64. To determine whether F proteins are functionally analogous to GP64 proteins, we deleted the
gp64
gene from an Ac
M
NPV bacmid and inserted F protein genes from three different baculoviruses. In addition, we also inserted envelope protein genes from vesicular stomatitis virus (VSV) and Thogoto virus. Transfection of the
gp64
-null bacmid DNA into Sf9 cells does not generate infectious particles, but this defect was rescued by introducing either the F protein gene from Ld
M
NPV or Se
M
NPV or the G protein gene from VSV. These results demonstrate that baculovirus F proteins are functionally analogous to GP64. Because baculovirus F proteins appear to be more widespread within the family and are much more divergent than GP64 proteins,
gp64
may represent the acquisition of an envelope protein gene by an ancestral baculovirus. The Ac
M
NPV pseudotyping system provides an efficient and powerful method for examining the functions and compatibilities of analogous or orthologous viral envelope proteins, and it could have important biotechnological applications.
Cellular Transcription Factor Sp1 Recruits Simian Virus 40 Capsid Proteins to the Viral Packaging Signal,
ses
Authors: Gordon-Shaag Ariela , Ben-Nun-Shaul Orly , Roitman Vered , Yosef Yael , Oppenheim Ariella
Published: July 2002

Simian virus 40 (SV40) capsid assembly occurs in the nucleus. All three capsid proteins bind DNA nonspecifically, raising the dilemma of how they attain specificity to the SV40 minichromosome in the presence of a large excess of genomic DNA. The SV40 packaging signal,
ses
, which is required for assembly, is composed of multiple DNA elements that bind transcription factor Sp1. Our previous studies showed that Sp1 participates in SV40 assembly and that it cooperates in DNA binding with VP2/3. We hypothesized that Sp1 recruits the capsid proteins to the viral minichromosome, conferring upon them specific DNA recognition. Here, we have tested the hypothesis. Computer analysis showed that the combination of six tandem GC boxes at
ses
is not found at cellular promoters and therefore is unique to SV40. Cooperativity in DNA binding between Sp1 and VP2/3 was not abolished at even a 1,000-fold excess of cellular DNA, providing strong support for the recruitment hypothesis. Sp1 also binds VP1 and cooperates with VP1 in DNA binding. VP1 pentamers (VP1
5
) avidly interact with VP2/3, utilizing the same VP2/3 domain as described for polyomavirus. We conclude that VP1
5
-VP2/3 building blocks are recruited by Sp1 to
ses
, where they form the nucleation center for capsid assembly. By this mechanism the virus ensures that capsid formation is initiated at a single site around its minichromosome. Sp1 enhances the formation of SV40 pseudovirions in vitro, providing additional support for the model. Analyses of Sp1 and VP3 deletion mutants showed that Sp1 and VP2/3 bind one another and cooperate in DNA binding through their DNA-binding domains, with additional contacts outside these domains. VP1 contacts Sp1 at residues outside the Sp1 DNA-binding domain. These and additional data allowed us to propose a molecular model for the VP1
5
-VP2/3-DNA-Sp1 complex.
Characterization of the Arenavirus RING Finger Z Protein Regions Required for Z-Mediated Inhibition of Viral RNA Synthesis
Authors: Cornu Tatjana I. , de la Torre Juan Carlos
Published: July 2002

The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an enveloped virus with a bisegmented negative-strand RNA genome whose proteomic capability is limited to four polypeptides, namely, nucleoprotein; surface glycoprotein (GP), which is proteolytically processed into GP1 and GP2; polymerase (L); and a small (11-kDa) RING finger protein (Z). Using a reverse genetic system based on the ARM strain of LCMV, we have previously shown that Z has a strong inhibitory activity on LCMV minigenome transcription and RNA replication (T. I. Cornu and J. C. de la Torre, J. Virol. 75:9415-9426, 2001). In the present study, we have identified regions and specific amino acid residues within Z which contribute to its inhibitory activity on RNA synthesis mediated by the LCMV polymerase. Z proteins from different LCMV strains had similar inhibitory activities on the expression of the LCMV ARM minigenome, whereas the Z protein of the genetically more distantly related Tacaribe virus had an approximately 10-fold lower inhibitory activity on ARM minigenome expression. Results from the use of chimera proteins between Z and
Xenopus
Neuralized, a nonviral RING finger protein, indicated that the structural integrity of the Z RING domain (RD) was required but not sufficient for the inhibitory activity of Z. Serial deletion mutants of the N and C termini of Z showed that the N terminus (residues 1 through 16) and C terminus (residues 79 through 90) do not contribute to the Z inhibitory activity. A highly conserved tryptophan (W) residue located at position 36 in ARM-Z, next to the second conserved cysteine (C) residue of the Z RD, also contributed to the Z inhibitory activity.
Interleukin-1β Expression in Human Gastric Carcinoma with Epstein-Barr Virus Infection
Authors: Chong Ja-Mun , Sakuma Kazuya , Sudo Makoto , Osawa Toshio , Ohara Etsuko , Uozaki Hiroshi , Shibahara Junji , Kuroiwa Kenji , Tominaga Shin-ichi , Hippo Yoshitaka , Aburatani Hiroyuki , Funata Nobuaki , Fukayama Masashi
Published: July 2002

The KT tumor is a transplantable strain of a human Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), established in severe combined immunodeficiency (SCID) mice, with which the cytokine expression of EBVaGC can be investigated without interference from the infiltrating lymphocytes. As a part of a high-density oligonucleotide array (GeneChip) analysis of EBVaGC, the interleukin-1β (IL-1β) gene was the only cytokine gene that showed markedly higher expression in the KT tumor cells than in two tumor strains of EBV-negative GC. The results were confirmed by Northern blotting, Western blotting, and enzyme-linked immunosorbent assay. Furthermore, we demonstrated a positive signal for IL-1β mRNA in the carcinoma cells of a surgically resected EBVaGC, but not in EBV-negative GC, by in situ hybridization. In vitro, IL-1β increased the cell growth of a GC cell line, TMK1. Thus, IL-1β may act as an autocrine growth factor in EBVaGC.
Bacterial Superantigen Exposure after Resolution of Influenza Virus Infection Perturbs the Virus-Specific Memory CD8+-T-Cell Repertoire
Authors: Huang Chiu-Chen , Shah Sangeeta , Nguyen Phuong , Altman John D. , Blackman Marcia A.
Published: July 2002

Heterologous viral infections have been shown to impact the preexisting memory CD8+-T-cell repertoire. Bacterial superantigens are products of common human pathogenic bacteria, including staphylococci and streptococci, that are potent T-cell-stimulatory molecules. In this report, we show that exposure to staphylococcal enterotoxin B, a bacterial superantigen, causes a selective functional deletion of cross-reactive influenza virus-specific CD8+memory T cells. This perturbation of the memory repertoire can have a significant impact on viral clearance after secondary challenge.
Selection for c-mycIntegration Sites in Polyclonal T-Cell Lymphomas
Authors: Broussard Dana R. , Mertz Jennifer A. , Lozano M. , Dudley Jaquelin P.
Published: July 2002

Type B leukemogenic virus (TBLV) is highly related to mouse mammary tumor virus but induces rapidly appearing T-cell lymphomas in mice. Unlike other T-cell tumors induced by retroviruses, only 5 to 10% of TBLV-induced lymphomas have detectable viral integrations near c-mycby Southern blotting, whereas Northern blotting has shown that most tumors have two- to sixfold overexpression of c-mycRNA. In this report, PCR was used to demonstrate that at least 30% of these lymphomas have TBLV insertions near c-myc. Some tumors contained multiple TBLV proviruses in different locations and orientations, suggesting that the tumors are polyclonal. The integrated proviruses near c-mychad different numbers (two to four) of long terminal repeat (LTR) enhancer repeats, although LTRs with three-repeat enhancers dominated the proviral population. Passage of polyclonal tumors in immunocompetent mice and semiquantitative PCR revealed that only cells with particular integrations were selected for growth. In three of six tumors tested, proviruses containing four-repeat enhancers near c-mycwere selected during tumor passage. Since tumor cell selection may be accomplished by overexpression of c-mycRNA due to proximity to the unique TBLV LTR enhancer, we inserted LTRs at various locations within a plasmid containing the entire c-myclocus and cellular flanking sequences. To quantitatively measure effects on transcription, theRenillaluciferase gene was substituted for most of c-mycexon 2, and transient transfections were performed with c-mycreporter constructs in two different T-cell lines. As expected, insertion of a TBLV LTR with three-repeat enhancers in either orientation, 5" and 3", of themycgene elevated reporter activity from 2- to 160-fold, consistent with enhancer function, but four-repeat LTRs had lower levels of expression compared to three-repeat LTRs. Surprisingly, LTR insertions that gave maximal c-mycexpression in transient-transfection assays declined in tumor cells selected for growth in vivo. Selection for clonal growth may occur in tumor cells that have modest c-mycoverexpression after proviral insertion to prevent apoptosis.
The Human Cytomegalovirus UL35 Gene Encodes Two Proteins with Different Functions
Authors: Liu Yingguang , Biegalke Bonita J.
Published: July 2002

The human cytomegalovirus (HCMV) virion is a complex structure that contains at least 30 proteins, many of which have been identified. We determined that the HCMV UL35 gene encodes two proteins, including a previously unidentified virion protein. A 22-kDa phosphoprotein (ppUL35
A
) was translated from a 1.2-kb UL35 transcript by 4 h postinfection; a second phosphoprotein of 75 kDa (ppUL35) was translated from a 2.2-kb transcript predominantly late in infection. The 22-kDa protein localized to the nucleus, while the 75-kDa protein localized to the juxtanuclear compartment and was packaged into virion particles. The 22-kDa protein was identical to the COOH-terminal end of the 75-kDa protein but was not found in virions, thus defining the NH
2
-terminal portion of the 75-kDa protein as essential for packaging. Expression of the 22-kDa protein inhibited activation of the major immediate-early promoter by ppUL82 (pp71), suggesting that the UL35 22-kDa protein may modulate expression of the major immediate-early gene.
Herpes Simplex Virus Selectively Induces Expression of the CC Chemokine RANTES/CCL5 in Macrophages through a Mechanism Dependent on PKR and ICP0
Authors: Melchjorsen Jesper , Pedersen Finn S. , Mogensen Søren C. , Paludan Søren R.
Published: July 2002

Recruitment of leukocytes is essential for eventual control of virus infections. Macrophages represent a leukocyte population involved in the first line of defense against many infections, including herpes simplex virus (HSV) infection. Through presentation of antigens to T cells and production of cytokines and chemokines, macrophages also constitute an important link between the innate and adaptive immune systems. Here, we have investigated the chemokine expression profile of macrophages after HSV infection and the virus-cell interactions involved. By reverse transcription-PCR and cDNA arrays, we found that HSV type 1 (HSV-1) and HSV-2 induced expression of the CC chemokine RANTES/CCL5 in murine macrophage cell lines and peritoneal cells. The CXC chemokine BCA-1/CXCL13 was also induced in peritoneal cells. Twenty-six other chemokines tested were not affected. Accumulation of RANTES mRNA was detectable after 5 h of infection, was sensitive to UV irradiation of the virus, and was preceded by accumulation of viral immediate-early mRNA and proteins. The viral components responsible for initiation of RANTES expression were examined with virus mutants and RAW 264.7 macrophage-like cells expressing a dominant negative mutant of the double-stranded-RNA-activated protein kinase (PKR). The PKR mutant cell line displayed reduced constitutive and HSV-inducible RANTES expression compared to the control cell line. HSV-1 mutants deficient in genes encoding the immediate-early proteins ICP4, ICP22, and ICP27 remained fully capable of inducing RANTES expression in macrophages. By contrast, the ability of an ICP0-deficient HSV-1 mutant to induce RANTES expression was compromised. Thus, HSV selectively induces expression of RANTES in macrophages through a mechanism dependent on cellular PKR and viral ICP0.
Capsid Protein C of Tick-Borne Encephalitis Virus Tolerates Large Internal Deletions and Is a Favorable Target for Attenuation of Virulence
Authors: Kofler Regina M. , Heinz Franz X. , Mandl Christian W.
Published: July 2002

Deletions ranging in size from 4 to 21 amino acid residues were introduced into the capsid protein of the flavivirus tick-borne encephalitis (TBE) virus. These deletions incrementally affected a hydrophobic domain which is present at the center of all flavivirus capsid protein sequences and part of which may form an amphipathic alpha-helix. In the context of the full-length TBE genome, the deletions did not measurably affect protein expression and up to a deletion length of 16 amino acid residues, corresponding to almost 17% of mature protein C, viable virus was recovered. This virus was strongly attenuated but highly immunogenic in adult mice, revealing capsid protein C as a new and attractive target for the directed attenuation of flaviviruses. Apparently, the larger deletions interfered with the correct assembly of infectious virus particles, and this disturbance of virion assembly is likely to be the molecular basis of attenuation. However, all of the mutants carrying large deletions produced substantial amounts of subviral particles, which as judged from density gradient analyses were identical to recombinant subviral particles as obtained by the expression of the surface proteins prM and E alone. The structural and functional flexibility of protein C revealed in this study and its predicted largely alpha-helical conformation are reminiscent of capsid proteins of other enveloped viruses, such as alphaviruses (N-terminal domain of the capsid protein), retroviruses, and hepadnaviruses and suggest that all of these may belong to a common structural class, which is fundamentally distinct from the classical β-barrel structures of many icosahedral viral capsids. The possibility of attenuating flaviviruses by disturbing virus assembly and favoring the production of noninfectious but highly immunogenic subviral particles opens up a promising new avenue for the development of live flavivirus vaccines.
Preferential Localization of the Epstein-Barr Virus (EBV) Oncoprotein LMP-1 to Nuclei in Human T Cells: Implications for Its Role in the Development of EBV Genome-Positive T-Cell Lymphomas
Authors: Xu Jingwu , Ahmad Ali , Menezes José
Published: July 2002

The Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP-1) is thought to play a role in the EBV-induced B-cell transformation and immortalization. EBV has also been implicated in certain human T-cell lymphomas; however, the phenotypic effects of the expression of this oncoprotein in T cells are not known. To learn whether LMP-1 also induces phenotypic changes in T cells, we stably expressed it in human cell lines of T and B lineages and 25 LMP-1-expressing T-cell clones and 7 B-cell clones were examined. Our results show for the first time that, in sharp contrast to B cells, LMP-1 preferentially localizes to nuclei in T cells and does not induce the phenotypic changes in these cells that it induces in B cells, does not associate with TRAF proteins, and does not arrest the cell cycle in the G2/M phase. A computer-assisted analysis revealed that LMP-1 lacks the canonical nuclear localization signal. Our results suggest that this oncoprotein may not play the same role in the lymphomagenesis of T cells as it does in B cells.
Induction of Long-Term Memory CD8+T Cells for Recall of Viral Clearing Responses against Influenza Virus
Authors: Deliyannis Georgia , Jackson David C. , Ede Nicholas J. , Zeng Weiguang , Hourdakis Irene , Sakabetis Evaline , Brown Lorena E.
Published: July 2002

Induction of cytotoxic T-cell-mediated virus-clearing responses by influenza virus T cell determinant-containing peptide immunogens was examined. The most potent synthetic immunogens for eliciting pulmonary viral-clearing responses contained peptides representing determinants for CD4 and CD8 T cells (TH and CTL peptides, respectively) together with two or four palmitic acid (Pal) groups. Inoculated in adjuvant, these Pal2- or Pal4-CTL-TH lipopeptides and the nonlipidated CTL peptide induced equivalent levels of cytolytic activity in the primary effector phase of the response. The ability to recall lytic responses, however, diminished much more rapidly in CTL peptide-primed than in lipopeptide-primed mice. By 15 months postpriming, the recalled lytic activity in lipopeptide-inoculated mice remained potent, but the response induced by the CTL peptide was weak. Enumeration of specific gamma interferon-secreting CD8 T cells revealed that a greater number of these T cells had entered or remained in the memory pool in lipopeptide-primed mice, arguing for a quantitative rather than qualitative enhancement of the response on recall. Addition of either the lipid or the TH peptide to the CTL peptide was not sufficient to provide these long-lived antiviral responses, but inclusion of both components augmented the response. CD4 T cells elicited by the lipopeptides did not influence the rate of viral clearance upon challenge and most likely had a role in induction or maintenance of the memory response. It therefore appears that the lipopeptide immunogens, although not significantly superior at inducing primary effector CD8 T cells, elicit a much more effective memory population, the recall of which may account for their superiority in inducing pulmonary protection after viral challenge.
Specific Incorporation of Heat Shock Protein 70 Family Members into Primate Lentiviral Virions
Authors: Gurer Cagan , Cimarelli Andrea , Luban Jeremy
Published: July 2002

To determine if any heat shock proteins are incorporated into human immunodeficiency virus type 1 (HIV-1) virions in a manner similar to that of the peptidyl-prolyl isomerase cyclophilin A, we probed purified virions with antibodies against heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70, Hsc70, and Hsp90. Of these proteins, Hsp60, Hsp70, and Hsc70 associated with virions purified based on either particle density or size and were shown to be incorporated within the virion membrane, where they were protected from digestion by exogenous protease. Virion incorporation of Hsp70 was also observed with HIV-2 and with simian immunodeficiency viruses SIV
MAC
and SIV
AGM
, but it appears to be specific for primate lentiviruses, since Hsp70 was not detected in association with Moloney murine leukemia virus virions. Of the HIV-1 genes,
gag
was found to be sufficient for Hsp70 incorporation, though Hsp70 was roughly equimolar with
pol
-encoded proteins in virions.
Interaction of Mn(III)Tetraphenylporphyrin with Superoxide; The Reaction Mechanism and Evidence for a Peroxo Complex
Authors: Lang Kamil , Vondrák Jiří , Wagnerová Dana M.
Published: July 2002

Cathodic one-electron reduction of Mn(III)TPP and O2 in an aprotic solvent yields Mn(II)TPP and the superoxide anion radical O2-. The Mn(II)TPP and O2- react in solution to the side-on peroxo complex Mn(III)TPP-O22-, which is oxidized at the electrode at a peak potential of Epa = 0.40 V vs NHE (k ≈ 104 dm3 mol-1 s-1). The rate-determining step of the overall two-electron oxidation is the transfer of the first electron coupled with a structure rearrangement of the peroxo complex to a superoxo complex.
Hydration Analysis Based Calculation of Solute Activity Coefficients in Ternary Saturated Solutions
Authors: Eysseltová Jitka
Published: July 2002

Based on recently developed hydration analysis of solubility isotherms, a method for calculating activity coefficients of solute in ternary saturated solution is suggested. The method needs no adjustable parameters; it takes only solubility data and the value of the activity coefficient of the respective solute in its binary saturated solution under identical conditions as starting point. In the NaCl - KCl - H2O model system, a comparison between the calculated activity coefficients and those obtained by de Lima and Pitzer from solution properties and from the solubility shows a good agreement over a wide temperature range.
Study of Sorption of Triphenylmethane Dyes on a Magnetic Carrier Bearing an Immobilized Copper Phthalocyanine Dye
Authors: Šafařík Ivo , Šafaříková Miroslava , Vrchotová Naděžda
Published: July 2002

Magnetite particles bearing covalently immobilized copper phthalocyanine dye ("blue magnetite") were prepared and used for the sorption of triphenylmethane dyes from aqueous solutions. The binding of some triphenylmethane dyes bearing two or three amino groups (basic fuchsin, crystal violet, malachite green) followed the Langmuir adsorption model. The maximum adsorption capacities were calculated. Dyes having no amino group in their molecules exhibited only low adsorption to immobilized copper phthalocyanine. The presence of amino groups in the molecules of triphenylmethane dyes seems to be necessary for their efficient binding on blue magnetite. Generally, efficient sorption of the organic compounds to immobilized copper phthalocyanine may be conditional on a cooperative effect of two parameters, viz. a certain planarity of the sorbate molecule and interaction of the central copper ion of the phthalocyanine molecule with the sorbate amino group or heterocyclic nitrogen atom.
On Some 2,4,6-Triphenylpyridinium 2-Benzimidazolate Inner Salts
Authors: Böhm Stanislav , Ruppertová Iveta , Havlíček Vladimír , Kuthan Josef
Published: July 2002

Molecular and electronic structure of the titled inner salts 1a-1c is discussed on the basis of the PM3, AM1 and CNDO/2 calculations. Compounds 1a, 1b and 1c were prepared from 1-(benzimidazol-2-yl)-2,6-diphenyl-4-(4-X-phenyl)pyridinium perchlorates (2a-2c) by chromatography on alumina only in forms of relatively stable clusters with water and solvent molecules. Mass spectra of the prepared substances are discussed.
Functionalization of Lupane with Chromium(VI) Oxide. A Remark on the Structure of Clerodone
Authors: Sejbal Jan , Klinot Jiří , Buděšínský Miloš
Published: July 2002

The reaction of lupane (1) with chromium oxide gave lup-2-en-1-one (3), lupan-12-one (2), lupan-16-one (isolated after hydride reduction as lupan-16α-ol (4)), and lup-18-en-21-one (5) in low yields. The same oxidation of 3β,28-dimethoxylupane (11) afforded no products of functionalization in non-activated positions; only products of oxidation of the 3β-methoxy group and of the oxidation cleavage of the A ring were obtained, viz. 28-methoxylupan-3β-ol formate (12), 28-methoxylupan-3-one (14), and 28-methoxy-2-norlupane-1,3-dioic anhydride (13). The structure of the compounds obtained was confirmed by their 1H NMR, 13C NMR, and mass spectra. The lupan-12-one (2) prepared is not identical with clerodone, whose isolation from Clerodendron infortunatum BHAT. was described in 1965 and to which the structure of lupan-12-one was then ascribed.