bluuebunny/crossref_metadata_2025 huggingface dataset:
Adeno-Associated Virus Type 2-Mediated Gene Transfer: Role of Cellular FKBP52 Protein in Transgene Expression
Authors: Qing Keyun , Hansen Jonathan , Weigel-Kelley Kirsten A. , Tan Mengqun , Zhou Shangzhen , Srivastava Arun
Published: July 2002

Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful vector for human gene therapy, the transduction efficiencies of AAV vectors vary greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays a crucial role in AAV-mediated transgene expression (K. Y. Qing, X.-S. Wang, D. M. Kube, S. Ponnazhagan, A. Bajpai, and A. Srivastava, Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997). We have documented a strong correlation between the phosphorylation state of ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing, B. Khuntrirat, C. Mah, D. M. Kube, X.-S. Wang, S. Ponnazhagan, S. Z. Zhou, V. J. Dwarki, M. C. Yoder, and A. Srivastava, J. Virol
.
72:1593–1599, 1998). We have also established that the ssD-BP is phosphorylated by epidermal growth factor receptor protein tyrosine kinase and that the tyrosine-phosphorylated form, but not the dephosphorylated form, of ssD-BP prevents AAV second-strand DNA synthesis and, consequently, results in a significant inhibition of AAV-mediated transgene expression (C. Mah, K. Y. Qing, B. Khuntrirat, S. Ponnazhagan, X.-S. Wang, D. M. Kube, M. C. Yoder, and A. Srivastava, J. Virol
.
72:9835–9841, 1998). Here, we report that a partial amino acid sequence of ssD-BP purified from HeLa cells is identical to a portion of a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein 52 (FKBP52). FKBP52 was purified by using a prokaryotic expression plasmid containing the human cDNA. The purified protein could be phosphorylated at both tyrosine and serine or threonine residues, and only the phosphorylated forms of FKBP52 were shown to interact with the AAV single-stranded D-sequence probe. Furthermore, in in vitro DNA replication assays, tyrosine-phosphorylated FKBP52 inhibited AAV second-strand DNA synthesis by greater than 90%. Serine- or threonine-phosphorylated FKBP52 caused ≈40% inhibition, whereas dephosphorylated FKBP52 had no effect on AAV second-strand DNA synthesis. Deliberate overexpression of FKBP52 effectively reduced the extent of tyrosine phosphorylation of the protein, resulting in a significant increase in AAV-mediated transgene expression in human and murine cell lines. These studies corroborate the idea that the phosphorylation status of the cellular FKBP52 protein correlates strongly with AAV transduction efficiency, which may have important implications for the optimal use of AAV vectors in human gene therapy.
Macrophage Tropism of Human Immunodeficiency Virus Type 1 Isolates from Brain and Lymphoid Tissues Predicts Neurotropism Independent of Coreceptor Specificity
Authors: Gorry Paul R. , Bristol Greg , Zack Jerome A. , Ritola Kimberly , Swanstrom Ronald , Birch Chris J. , Bell Jeanne E. , Bannert Norbert , Crawford Keith , Wang Hui , Schols Dominique , De Clercq Erik , Kunstman Kevin , Wolinsky Steven M. , Gabuzda Dana
Published: July 2002

The viral determinants that underlie human immunodeficiency virus type 1 (HIV-1) neurotropism are unknown, due in part to limited studies on viruses isolated from brain. Previous studies suggest that brain-derived viruses are macrophage tropic (M-tropic) and principally use CCR5 for virus entry. To better understand HIV-1 neurotropism, we isolated primary viruses from autopsy brain, cerebral spinal fluid, blood, spleen, and lymph node samples from AIDS patients with dementia and HIV-1 encephalitis. Isolates were characterized to determine coreceptor usage and replication capacity in peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDM), and microglia. Env V1/V2 and V3 heteroduplex tracking assay and sequence analyses were performed to characterize distinct variants in viral quasispecies. Viruses isolated from brain, which consisted of variants that were distinct from those in lymphoid tissues, used CCR5 (R5), CXCR4 (X4), or both coreceptors (R5X4). Minor usage of CCR2b, CCR3, CCR8, and Apj was also observed. Primary brain and lymphoid isolates that replicated to high levels in MDM showed a similar capacity to replicate in microglia. Six of 11 R5 isolates that replicated efficiently in PBMC could not replicate in MDM or microglia due to a block in virus entry. CD4 overexpression in microglia transduced with retroviral vectors had no effect on the restricted replication of these virus strains. Furthermore, infection of transfected cells expressing different amounts of CD4 or CCR5 with M-tropic and non-M-tropic R5 isolates revealed a similar dependence on CD4 and CCR5 levels for entry, suggesting that the entry block was not due to low levels of either receptor. Studies using TAK-779 and AMD3100 showed that two highly M-tropic isolates entered microglia primarily via CXCR4. These results suggest that HIV-1 tropism for macrophages and microglia is restricted at the entry level by a mechanism independent of coreceptor specificity. These findings provide evidence that M-tropism rather than CCR5 usage predicts HIV-1 neurotropism.
CD8+T Lymphocytes Mediate Borna Disease Virus-Induced Immunopathology Independently of Perforin
Authors: Hausmann Jürgen , Schamel Karin , Staeheli Peter
Published: July 2002

Perforin-mediated lysis of target cells is the major antiviral effector mechanism of CD8+T lymphocytes. We have analyzed the role of perforin in a mouse model for CD8+T-cell-mediated central nervous system (CNS) immunopathology induced by Borna disease virus. When a defective perforin gene was introduced into the genetic background of the Borna disease-susceptible mouse strain MRL, the resulting perforin-deficient mice developed strong neurological disease in response to infection indistinguishable from that of their perforin-expressing littermates. The onset of disease was slightly delayed. Brains of diseased perforin-deficient mice showed similar amounts and a similar distribution of CD8+T cells as wild-type animals. Perforin deficiency had no impact on the kinetics of viral spread through the CNS. Unlike brain lymphocytes from diseased wild-type mice, lymphocytes from perforin-deficient MRL mice showed no in vitro cytolytic activity towards target cells expressing the nucleoprotein of Borna disease virus. Taken together, these results demonstrate that CD8+T cells mediate Borna disease independent of perforin. They further suggest that the pathogenic potential of CNS-infiltrating CD8+T cells does not primarily reside in their lytic activity but rather in other functions.
N-Terminal Cleavage Fragment of Glycosylated Gag Is Incorporated into Murine Oncornavirus Particles
Authors: Fujisawa Ryuichi , McAtee Frank J. , Favara Cynthia , Hayes Stanley F. , Portis John L.
Published: July 2002

Glycosylated Gag (Glycogag) is a transmembrane protein encoded by murine and feline oncornaviruses. While the protein is dispensible for virus replication, Glycogag-null mutants of a neurovirulent murine oncornavirus are slow to spread in vivo and exhibit a loss of pathogenicity. The function of this protein in the virus life cycle, however, is not understood. Glycogag is expressed at the plasma membrane of infected cells but has not been detected in virions. In the present study we have reexamined this issue and have found an N-terminal cleavage fragment of Glycogag which was pelleted by high-speed centrifugation and sedimented in sucrose density gradients at the same bouyant density as virus particles. Its association with virions was confirmed by velocity sedimentation through iodixanol, which effectively separated membrane microvesicles from virus particles. Furthermore, the apparent molecular weight of the virion-associated protein was different from that of the protein extracted from the plasma membrane, suggesting some level of specificity or selectivity of incorporation.
Open Reading Frame III of Borna Disease Virus Encodes a Nonglycosylated Matrix Protein
Authors: Kraus Ina , Eickmann Markus , Kiermayer Simone , Scheffczik Hanno , Fluess Manuela , Richt Jürgen A. , Garten Wolfgang
Published: July 2002

The open reading frame III of Borna disease virus (BDV) codes for a protein with a mass of 16 kDa, named p16 or BDV-M. p16 was described as an N-glycosylated protein in several previous publications and therefore was termed gp18, although the amino acid sequence of p16 does not contain any regular consensus sequence for N glycosylation. We examined glycosylation of p16 and studied its membrane topology using antisera raised against peptides, which comprise the N and the C termini. Neither an N- nor a C-terminal peptide is cleaved from p16 during maturation. Neither deglycosylation of p16 by endoglycosidases nor binding of lectin to p16 was detectable. Introduction of typical N-glycosylation sites at the proposed sites of p16 failed in carbohydrate attachment. Flotation experiments with membranes of BDV-infected cells on density gradients revealed that p16 is not an integral membrane protein, since it can be dissociated from membranes. Our experimental data strongly suggest that p16 is a typical nonglycosylated matrix protein associated at the inner surface of the viral membrane, as is true for homologous proteins of other members of the
Mononegavirales
order.
Mutations in Hepatitis C Virus RNAs Conferring Cell Culture Adaptation
Authors: Lohmann Volker , Körner Frank , Dobierzewska Aneta , Bartenschlager Ralf
Published: July 2002

As an initial approach to studying the molecular replication mechanisms of hepatitis C virus (HCV), a major causative agent of acute and chronic liver disease, we have recently developed selectable self-replicating RNAs. These replicons lacked the region encoding the structural proteins and instead carried the gene encoding the neomycin phosphotransferase. Although the replication levels of these RNAs within selected cells were high, the number of G418-resistant colonies was reproducibly low. In a search for the reason, we performed a detailed analysis of replicating HCV RNAs and identified several adaptive mutations enhancing the efficiency of colony formation by several orders of magnitude. Adaptive mutations were found in nearly every nonstructural protein but not in the 5′ or 3′ nontranslated regions. The most drastic effect was found with a single-amino-acid substitution in NS5B, increasing the number of colonies ∼500-fold. This mutation was conserved with RNAs isolated from one cell line, in contrast to other amino acid substitutions enhancing the efficiency of colony formation to a much lesser extent. Interestingly, some combinations of these nonconserved mutations with the highly adaptive one reduced the efficiency of colony formation drastically, suggesting that some adaptive mutations are not compatible.
Rapid Assessment of Adenovirus Serum Neutralizing Antibody Titer Based on Quantitative, Morphometric Evaluation of Capsid Binding and Intracellular Trafficking: Population Analysis of Adenovirus Capsid Association with Cells Is Predictive of Adenovirus Infectivity
Authors: Vincent Theresa , Harvey Ben-Gary , Hogan Suzanne M. , Bailey Christopher J. , Crystal Ronald G. , Leopold Philip L.
Published: July 2002

Neutralizing antiviral antibodies are typically detected on the basis of inhibition of viral function, such as propagation of a viral infection or inhibition of viral gene expression. Evidence is presented that anti-adenovirus neutralizing antibodies can be evaluated by analysis of cell-associated capsids or by analysis of intracellular trafficking of the capsids within 1 h after infection. Quantitative analyses of these morphologic parameters represent rapid, broadly applicable, functional assays for the detection of anti-adenovirus neutralizing antibodies.
Reactivation of the Human Cytomegalovirus Major Immediate-Early Regulatory Region and Viral Replication in Embryonal NTera2 Cells: Role of Trichostatin A, Retinoic Acid, and Deletion of the 21-Base-Pair Repeats and Modulator
Authors: Meier Jeffery L.
Published: July 2002

Inactivity of the human cytomegalovirus (HCMV) major immediate-early regulatory region (MIERR), which is composed of promoter, enhancer, unique region, and modulator, is linked to lack of HCMV replication in latently infected cells and in other nonpermissive cell types, including human embryonal NTera2 carcinoma (NT2) cells. I refined the embryonal NT2 cell model to enable characterization of the unknown mechanistic basis for silencing of HCMV MIERR-dependent transcription and viral replication in nonpermissive cells. These infected NT2 cells contain nonreplicating viral genomes with electrophoretic mobility equivalent to a supercoiled, bacterial artificial chromosome of comparable molecular weight. MIERR-dependent transcription is minimal to negligible. Increasing the availability of positive-acting transcription factors by retinoic acid (RA) treatment after infection is largely insufficient in reactivating the MIERR. In contrast, trichostatin A (TSA), a histone deacetylase inhibitor, reactivates MIERR-dependent transcription. Contrary to prior findings produced from transfected MIERR segments, deletion of the 21-bp repeats and modulator from the MIERR in the viral genome does not relieve MIERR silencing. To demonstrate that MIERR silencing likely results from enhancer inactivity, I examined an HCMV with a heterologous MIERR promoter that is enhancer dependent but exempt from IE2 p86-mediated negative autoregulation. This heterologous promoter, like its neighboring native MIERR promoter, exhibits immediate-early transcriptional kinetics in fibroblasts. In embryonal NT2 cells, the heterologous MIERR promoter is transcriptionally inactive. This silence is relieved by TSA but not by RA. Remarkably, TSA-induced reactivation of MIERR-dependent transcription from quiescent viral genomes is followed by release of infectious virus. I conclude that a mechanism of active repression imposes a block to MIERR-dependent transcription and viral replication in embryonal NT2 cells. Because TSA overcomes the block, viral gene silencing may involve histone deacetylase-based modification of viral chromatin, which might account for the covalently closed circular conformation of quiescent HCMV genomes.
Evidence of Infection with Simian Type D Retrovirus in Persons Occupationally Exposed to Nonhuman Primates
Authors: Lerche Nicholas W. , Switzer William M. , Yee JoAnn L. , Shanmugam Vedapuri , Rosenthal Ann N. , Chapman Louisa E. , Folks Thomas M. , Heneine Walid
Published: July 2002

Simian type D retrovirus (SRV) is enzootic in many populations of Asian monkeys of the genus
Macaca
and is associated with immunodeficiency diseases. However, the zoonotic potential of this agent has not been well defined. Screening for antibodies to SRV was performed as part of an ongoing study looking for evidence of infection with simian retroviruses among persons occupationally exposed to nonhuman primates (NHPs). Of 231 persons tested, 2 (0.9%) were found to be strongly seropositive, showing reactivity against multiple SRV antigens representing
gag
,
pol
, and
env
gene products by Western immunoblotting. Persistent long-standing seropositivity, as well as neutralizing antibody specific to SRV type 2, was documented in one individual (subject 1), while waning antibody with eventual seroreversion was observed in a second (subject 2). Repeated attempts to detect SRV by isolation in tissue culture and by using sensitive PCR assays for amplification of two SRV gene regions (
gag
and
pol
) were negative. Both individuals remain apparently healthy. We were also unable to transmit this seropositivity to an SRV-negative macaque by using inoculation of whole blood from subject 1. The results of this study provide evidence that occupational exposure to NHPs may increase the risk of infection with SRV and underscore the importance of both occupational safety practices and efforts to eliminate this virus from established macaque colonies.
Type D Retrovirus Gag Polyprotein Interacts with the Cytosolic Chaperonin TRiC
Authors: Hong Suntaek , Choi Gyu , Park Sunyoung , Chung An-Sik , Hunter Eric , Rhee Sung S.
Published: July 2002

The carboxy terminus-encoding portion of the
gag
gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1γ, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1γ also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.
Sequences at the 3′ Untranslated Region of Bamboo Mosaic Potexvirus RNA Interact with the Viral RNA-Dependent RNA Polymerase
Authors: Huang Cheng-Yen , Huang Yih-Leh , Meng Menghsiao , Hsu Yau-Heiu , Tsai Ching-Hsiu
Published: July 2002

The 3′ untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) genomic RNA was found to fold into a series of stem-loop structures including a pseudoknot structure. These structures were demonstrated to be important for viral RNA replication and were believed to be recognized by the replicase (C.-P. Cheng and C.-H. Tsai, J. Mol. Biol. 288:555–565, 1999). Electrophoretic mobility shift and competition assays have now been used to demonstrate that the
Escherichia coli
-expressed RNA-dependent RNA polymerase domain (Δ893) derived from BaMV open reading frame 1 could specifically bind to the 3′ UTR of BaMV RNA. No competition was observed when bovine liver tRNAs or poly(I)(C) double-stranded homopolymers were used as competitors, and the cucumber mosaic virus 3′ UTR was a less efficient competitor. Competition analysis with different regions of the BaMV 3′ UTR showed that Δ893 binds to at least two independent RNA binding sites, stem-loop D and the poly(A) tail. Footprinting analysis revealed that Δ893 could protect the sequences at loop D containing the potexviral conserved hexamer motif and part of the stem of domain D from chemical cleavage.
Purification and Characterization of West Nile Virus Nucleoside Triphosphatase (NTPase)/Helicase: Evidence for Dissociation of the NTPase and Helicase Activities of the Enzyme
Authors: Borowski Peter , Niebuhr Andreas , Mueller Oliver , Bretner Maria , Felczak Krzysztof , Kulikowski Tadeusz , Schmitz Herbert
Published: July 2002

The nucleoside triphosphatase (NTPase)/helicase associated with nonstructural protein 3 of West Nile (WN) virus was purified from cell culture medium harvested from virus-infected Vero cells. The purification procedure included sequential chromatography on Superdex-200 and Reactive Red 120 columns, followed by a concentration step on an Ultrogel hydroxyapatite column. The nature of the purified protein was confirmed by immunoblot analysis using a WN virus-positive antiserum, determination of its NH
2
terminus by microsequencing, and a binding assay with 5′-[
14
C]fluorosulfonylbenzoyladenosine. Under optimized reaction conditions the enzyme catalyzed the hydrolysis of ATP and the unwinding of the DNA duplex with
k
cat
values of 133 and 5.5 × 10
−3
s
−1
, respectively. Characterization of the NTPase activity of the WN virus enzyme revealed that optimum conditions with respect to the Mg
2+
requirement and the monovalent salt or polynucleotide response differed from those of other flavivirus NTPases. Initial kinetic studies demonstrated that the inhibition (or activation) of ATPase activity by ribavirin-5′-triphosphate is not directly related to changes in the helicase activity of the enzyme. Further analysis using guanine and
O
6
-benzoylguanine derivatives revealed that the ATPase activity of WN virus NTPase/helicase may be modulated, i.e., increased or reduced, with no effect on the helicase activity of the enzyme. On the other hand the helicase activity could be modulated without changing the ATPase activity. Our observations show that the number of ATP hydrolysis events per unwinding cycle is not a constant value.
Multiple Immediate-Early Gene-Deficient Herpes Simplex Virus Vectors Allowing Efficient Gene Delivery to Neurons in Culture and Widespread Gene Delivery to the Central Nervous System In Vivo
Authors: Lilley Caroline E. , Groutsi Filitsa , Han ZiQun , Palmer James A. , Anderson Patrick N. , Latchman David S. , Coffin Robert S.
Published: July 2002

Herpes simplex virus (HSV) has several potential advantages as a vector for delivering genes to the nervous system. The virus naturally infects and remains latent in neurons and has evolved the ability of highly efficient retrograde transport from the site of infection at the periphery to the site of latency in the spinal ganglia. HSV is a large virus, potentially allowing the insertion of multiple or very large transgenes. Furthermore, HSV does not integrate into the host chromosome, removing any potential for insertional activation or inactivation of cellular genes. However, the development of HSV vectors for the central nervous system that exploit these properties has been problematical. This has mainly been due to either vector toxicity or an inability to maintain transgene expression. Here we report the development of highly disabled versions of HSV-1 deleted for ICP27, ICP4, and ICP34.5/open reading frame P and with an inactivating mutation in VP16. These viruses express only minimal levels of any of the immediate-early genes in noncomplementing cells. Transgene expression is maintained for extended periods with promoter systems containing elements from the HSV latency-associated transcript promoter (J. A. Palmer et al., J. Virol. 74:5604–5618, 2000). Unlike less-disabled viruses, these vectors allow highly effective gene delivery both to neurons in culture and to the central nervous system in vivo. Gene delivery in vivo is further enhanced by the retrograde transport capabilities of HSV. Here the vector is efficiently transported from the site of inoculation to connected sites within the nervous system. This is demonstrated by gene delivery to both the striatum and substantia nigra following striatal inoculation; to the spinal cord, spinal ganglia, and brainstem following injection into the spinal cord; and to retinal ganglion neurons following injection into the superior colliculus and thalamus.
Rabies Virus-Based Vectors Expressing Human Immunodeficiency Virus Type 1 (HIV-1) Envelope Protein Induce a Strong, Cross-Reactive Cytotoxic T-Lymphocyte Response against Envelope Proteins from Different HIV-1 Isolates
Authors: McGettigan James P. , Foley Heather D. , Belyakov Igor M. , Berzofsky Jay A. , Pomerantz Roger J. , Schnell Matthias J.
Published: July 2002

Novel viral vectors that are able to induce both strong and long-lasting immune responses may be required as effective vaccines for human immunodeficiency virus type 1 (HIV-1) infection. Our previous experiments with a replication-competent vaccine strain-based rabies virus (RV) expressing HIV-1 envelope protein from a laboratory-adapted HIV-1 strain (NL4–3) and a primary HIV-1 isolate (89.6) showed that RV-based vectors are excellent for B-cell priming. Here we report that cytotoxic T-lymphocyte (CTL) responses against HIV-1 gp160 are induced by recombinant RVs. Our results indicated that a single inoculation of mice with an RV expressing HIV-1 gp160 induced a solid and long-lasting memory CTL response specific for HIV-1 envelope protein. Moreover, CTLs from immunized mice were not restricted to the homologous HIV-1 envelope protein and were able to cross-kill target cells expressing HIV-1 gp160 from heterologous HIV-1 strains. These studies further suggest promise for RV-based vectors to elicit a persistent immune response against HIV-1 and their potential utility as efficacious anti-HIV-1 vaccines.
Interleukin-1α Released from Epithelial Cells after Adenovirus Type 37 Infection Activates Intercellular Adhesion Molecule 1 Expression on Human Vascular Endothelial Cells
Authors: Chang Cheng-Hsien , Huang Yan , Issekutz Andrew C. , Griffith May , Lin Kuei-Hsiang , Anderson Robert
Published: July 2002

A key event in virus-induced inflammation (leukocyte extravasation through the endothelium) is the local activation of endothelial cells, as indicated by the expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin. In order to identify triggers of inflammation in adenovirus infection, we inoculated respiratory and ocular epithelial cells with adenovirus type 37 (Ad37), a human pathogen associated with keratoconjunctivitis as well as urogenital and respiratory infections. Fluids from virus-infected epithelial cells activated ICAM-1 (and to a lesser extent, VCAM-1) expression on cultured human umbilical vein endothelial cells. Blocking studies with anticytokine antibodies implicated interleukin-1α (IL-1α) as the epithelial cell-derived factor which activated endothelial cell ICAM-1 expression. The results thus identify epithelial cell-derived IL-1α as a potentially important activator of endothelial cells in Ad37-induced inflammation.
Envelope-Induced Cell Transformation by Ovine Betaretroviruses
Authors: Alberti Alberto , Murgia Claudio , Liu Shan-Lu , Mura Manuela , Cousens Chris , Sharp Mike , Miller A. Dusty , Palmarini Massimo
Published: July 2002

Ovine betaretroviruses include Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). JSRV and ENTV represent a unique class of oncogenic retroviruses that induce tumors of the respiratory tract. JSRV and ENTV are highly related but induce different diseases. Expression of the JSRV envelope (Env) induces transformation of rodent fibroblasts in vitro and phosphorylation of Akt, a central player in the phosphatidylinositol 3-kinase (PI-3K)/Akt signal transduction pathway. However, little information is available on the molecular biology of ENTV. In this study, we initially assessed whether the ENTV Env has the same properties as the homologous JSRV protein. We performed entry and interference assays using retroviral vectors pseudotyped with either the JSRV or the ENTV Env and sheep choroid plexus cells, choroid plexus cells stably expressing the JSRV Env protein, human 293T cells, mouse NIH 3T3 cells, or NIH 3T3 cells expressing human hyaluronidase 2 (HYAL2), the cellular receptor for JSRV. The results obtained indicated that ENTV and JSRV share the same receptor in sheep cells and that they can use human HYAL2 as a cellular receptor in mouse cells. The ENTV Env induces transformation of rodent fibroblasts in vitro. As with the JSRV Env, the tyrosine at position 590 is critical for ENTV Env-induced cell transformation, and Akt is phosphorylated in ENTV Env-transformed cells but not in the parental cell lines. Thus, ovine betaretroviruses share a common mechanism of cell transformation. We further investigated the relevance of Akt activation in cells transformed by ovine betaretroviruses. A PI-3K inhibitor blocked Akt phosphorylation in JSRV Env-transformed cells, suggesting a possible involvement of PI-3K in JSRV and ENTV Env-induced cell transformation. In addition, phosphorylated Akt was detected in a cell line derived from a lung tumor of a sheep with naturally occurring ovine pulmonary adenocarcinoma.
Alpha/Beta and Gamma Interferons Are Induced by Infection with Noncytopathic Bovine Viral Diarrhea Virus In Vivo
Authors: Charleston B. , Brackenbury L. S. , Carr B. V. , Fray M. D. , Hope J. C. , Howard C. J. , Morrison W. I.
Published: July 2002

In contrast to the results of previous in vitro studies, experimental infection of calves with noncytopathic bovine viral diarrhea virus (ncpBVDV) was found to induce strong alpha/beta and gamma interferon responses in gnotobiotic animals. These responses were associated with depressed levels of transforming growth factor β (TGF-β) in serum. The results of this study indicate that the immunosuppression caused by ncpBVDV is not associated with low interferon responses or elevated levels of TGF-β.
Cooperation between the Hemagglutinin of Avian Viruses and the Matrix Protein of Human Influenza A Viruses
Authors: Scholtissek Christoph , Stech Jürgen , Krauss Scott , Webster Robert G.
Published: July 2002

To analyze the compatibility of avian influenza A virus hemagglutinins (HAs) and human influenza A virus matrix (M) proteins M1 and M2, we doubly infected Madin-Darby canine kidney cells with amantadine (1-aminoadamantane hydrochloride)-resistant human viruses and amantadine-sensitive avian strains. By using antisera against the human virus HAs and amantadine, we selected reassortants containing the human virus M gene and the avian virus HA gene. In our system, high virus yields and large, well-defined plaques indicated that the avian HAs and the human M gene products could cooperate effectively; low virus yields and small, turbid plaques indicated that cooperation was poor. The M gene products are among the primary components that determine the species specificities of influenza A viruses. Therefore, our system also indicated whether the avian HA genes effectively reassorted into the genome and replaced the HA gene of the prevailing human influenza A viruses. Most of the avian HAs that we tested efficiently cooperated with the M gene products of the early human A/PR/8/34 (H1N1) virus; however, the avian HAs did not effectively cooperate with the most recently isolated human virus that we tested, A/Nanchang/933/95 (H3N2). Cooperation between the avian HAs and the M proteins of the human A/Singapore/57 (H2N2) virus was moderate. These results suggest that the currently prevailing human influenza A viruses might have lost their ability to undergo antigenic shift and therefore are unable to form new pandemic viruses that contain an avian HA, a finding that is of great interest for pandemic planning.
Complement-Mediated Enhancement of Antibody Function for Neutralization of Pseudotype Virus Containing Hepatitis C Virus E2 Chimeric Glycoprotein
Authors: Meyer Keith , Basu Arnab , Przysiecki Craig T. , Lagging L. Martin , Di Bisceglie Adrian M. , Conley Anthony J. , Ray Ranjit
Published: July 2002

We previously reported a number of features of hepatitis C virus (HCV) chimeric glycoproteins related to pseudotype virus entry into mammalian cells. In this study, pseudotype virus was neutralized by HCV E2 glycoprotein-specific antibodies and infected human sera. Neutralization (50% reduction of pseudotype virus plaque formation) was observed with two human immunoglobulin G1 monoclonal antibodies (MAbs) at concentrations of between 2.5 and 10 μg/ml. A hyperimmune rabbit antiserum to an E2 hypervariable region 1 (HVR1) mimotope also exhibited an HCV E2 pseudotype virus neutralization titer of ∼1/50. An E1 pseudotype virus used as a negative control was not neutralized to a significant level (<1/10) by these MAbs or rabbit antiserum to E2 HVR1. Since HCV probably has a lipid envelope, the role of complement in antibody-mediated virus neutralization was examined. Significant increases in the neutralization titers of the human MAbs (∼60- to 160-fold higher) and rabbit antiserum to HVR1 mimotopes (∼10-fold higher) were observed upon addition of guinea pig complement. Further, these studies suggested that complement activation occured primarily by the classical pathway, since a deficiency in the C4 component led to a significant decrease in the level of virus neutralization. This same decrease was not observed with factor B-deficient complement. We also determined that 9 of 56 HCV-infected patient sera (16%) had detectable pseudotype virus neutralization activity at serum dilutions of between 1/20 and 1/50 and that complement addition enhanced the neutralization activity of some of the HCV-infected human sera. Taken together, these results suggest that during infection, HCV E2 glycoprotein induces a weak neutralizing antibody response, that those antibodies can be measured in vitro by the surrogate pseudotype virus plaque reduction assay, and that neutralization function can be augmented by complement.
SUMO-1 Modification of Human Cytomegalovirus IE1/IE72
Authors: Spengler Mary L. , Kurapatwinski Karen , Black Adrian R. , Azizkhan-Clifford Jane
Published: July 2002

Human cytomegalovirus (HCMV) immediate-early protein IE1/IE72 is involved in undermining many cellular processes including cell cycle regulation, apoptosis, nuclear architecture, and gene expression. The multifunctional nature of IE72 suggests that posttranslational modifications may modulate its activities. IE72 is a phosphoprotein and has intrinsic kinase activity (S. Pajovic, E. L. Wong, A. R. Black, and J. C. Azizkhan, Mol. Cell. Biol. 17:6459-6464, 1997). We now demonstrate that IE72 is covalently conjugated to the small ubiquitin-like modifier (SUMO-1). SUMO-1 is an 11.5-kDa protein that is conjugated to multiple proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. A covalently modified protein migrating at ∼92 kDa, which is stabilized by a SUMO-1 hydrolase inhibitor, is revealed by Western blotting with anti-IE72 of lysates from cells infected with HCMV or cells expressing IE72. SUMO modification of IE72 was confirmed by immunoprecipitation with anti-IE72 and anti-SUMO-1 followed by Western blotting with anti-SUMO-1 and anti-IE72, respectively. Lysine 450 is within a sumoylation consensus site (I,V,L)KXE; changing lysine 450 to arginine by point mutation abolishes SUMO-1 modification of IE72. Inhibition of protein phosphatase 1 and 2A, which increases the phosphorylation of IE72, suppresses the formation of SUMO-1-IE72 conjugates. Both wild-type IE72 and IE72
K450R
localize to nuclear PML oncogenic domains and disrupt them. Studies of protein stability, transactivation, and complementation of IE72-deficient HCMV (CR208) have revealed no significant differences between wild-type IE72 and IE72
K450R
.
Selectable Subgenomic and Genome-Length Dicistronic RNAs Derived from an Infectious Molecular Clone of the HCV-N Strain of Hepatitis C Virus Replicate Efficiently in Cultured Huh7 Cells
Authors: Ikeda Masanori , Yi MinKyung , Li Kui , Lemon Stanley M.
Published: July 2002

Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 cells (Lohmann et al., Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et al., Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity-determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005→I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations.
The M184V Mutation Reduces the Selective Excision of Zidovudine 5′-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1
Authors: Boyer Paul L. , Sarafianos Stefan G. , Arnold Edward , Hughes Stephen H.
Published: July 2002

The M184V mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) causes resistance to lamivudine, but it also increases the sensitivity of the virus to zidovudine (3′-azido-3′-deoxythymidine; AZT). This sensitization to AZT is seen both in the presence and the absence of the mutations that confer resistance to AZT. AZT resistance is due to enhanced excision of AZT 5′-monophosphate (AZTMP) from the end of the primer by the RT of the resistant virus. Published data suggest that the excision reaction involves pyrophosphorolysis but that the likely in vivo pyrophosphate donor is not pyrophosphate but ATP. The mutations that lead to AZT resistance enhance ATP binding and, in so doing, enhance pyrophosphorolysis. The excision reaction is specific for AZT because HIV-1 RT, which can form a closed complex with a dideoxy-terminated primer and an incoming deoxynucleoside triphosphate (dNTP), does not form the closed complex with an AZTMP-terminated primer and an incoming dNTP. This means that an AZTMP-terminated primer has better access to the site where it can be excised. The M184V mutation alters the polymerase active site in a fashion that specifically interferes with ATP-mediated excision of AZTMP from the end of the primer strand. The M184V mutation does not affect the incorporation of AZT 5′-triphosphate (AZTTP), either in the presence or the absence of mutations that enhance AZTMP excision. However, in the presence of ATP, the M184V mutation does decrease the ability of HIV-1 RT to carry out AZTMP excision. Based on these results, and on the results of other excision experiments, we present a model to explain how the M184V mutation affects AZTMP excision.
Human T-Lymphotropic Virus Type 1 Oncoprotein Tax Promotes S-Phase Entry but Blocks Mitosis
Authors: Liang Min-Hui , Geisbert Thomas , Yao Yao , Hinrichs Steven H. , Giam Chou-Zen
Published: July 2002

Human T-lymphotropic virus type 1 (HTLV-1) Tax exerts pleiotropic effects on multiple cellular regulatory processes to bring about NF-κB activation, aberrant cell cycle progression, and cell transformation. Here we report that Tax stimulates cellular G
1
/S entry but blocks mitosis. Tax expression in naive cells transduced with a retroviral vector, pBabe-Tax, leads to a significant increase in the number of cells in the S phase, with an accompanying rise in the population of cells with a DNA content of 4N or more. In all cell types tested, including BHK-21, mouse NIH 3T3, and human diploid fibroblast WI-38, Tax causes an uncoupling of DNA synthesis from cell division, resulting in the formation of multinucleated giant cells and cells with decondensed, highly convoluted and lobulated nuclei that are reminiscent of the large lymphocytes with cleaved or cerebriform nuclei seen in HTLV-1-positive individuals. This contrasts with the Tax-transformed cell lines, PX1 (fibroblast) and MT4 (lymphocyte), which produce Tax at high levels, but without the accompanying late-stage cell cycle abnormalities. PX1 and MT4 may have been selected to harbor somatic mutations that allow a bypass of the Tax-induced block in mitosis.
New Insights into the Spring-Loaded Conformational Change of Influenza Virus Hemagglutinin
Authors: Gruenke Jennifer A. , Armstrong R. Todd , Newcomb William W. , Brown Jay C. , White Judith M.
Published: July 2002

Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix “spring-loaded” conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ∼50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.
The Oncogenic Protein Kinase Tpl-2/Cot Contributes to Epstein-Barr Virus-Encoded Latent Infection Membrane Protein 1-Induced NF-κB Signaling Downstream of TRAF2
Authors: Eliopoulos Aristides G. , Davies Clare , Blake Sarah S. M. , Murray Paul , Najafipour Sohrab , Tsichlis Philip N. , Young Lawrence S.
Published: July 2002

The Epstein-Barr virus-encoded latent infection membrane protein 1 (LMP1) is a pleiotropic protein, the activities of which include effects on cell transformation and phenotype, growth, and survival. The ability of LMP1 to mediate at least some of these phenomena could be attributed to the activation of the transcription factor NF-κB. LMP1 promotes NF-κB activation through the recruitment of the adapter protein TRAF2 and the formation of a dynamic multiprotein complex that includes the NF-κB kinase, the IκB kinases, and their downstream targets, IκBs and p105. In this study, we have identified the oncogenic kinase Tpl-2/Cot as a novel component of LMP1-induced NF-κB signaling. We show that Tpl-2 is expressed in primary biopsies from patients with nasopharyngeal carcinoma and Hodgkin's disease, where LMP1 is also found. Inducible expression of LMP1 promotes the activation of Tpl-2, and a catalytically inactive Tpl-2 mutant suppresses LMP1-induced NF-κB signaling. In colocalization and coimmunoprecipitation experiments, Tpl-2 and TRAF2 were found to interact with Tpl-2 functioning downstream of TRAF2. Consistent with this observation, catalytically inactive Tpl-2 also blocked CD40-mediated NF-κB activation, which largely depends on TRAF2. The ability of Tpl-2 to influence LMP1-induced NF-κB occurs through modulation of both IκBα and p105 functions. Furthermore, Tpl-2 was found to influence the expression of angiogenic mediators, such as COX-2 in LMP1-transfected cells. These data identify Tpl-2 as a component of LMP1 signaling downstream of TRAF2 and as a modulator of LMP1-mediated effects.
Chemometric Analysis of Substituent Effects. I. Substituent Effects on the Dissociation of Monosubstituted Benzoic Acids
Authors: Pytela Oldřich
Published: July 2002

Forty-six representative sets of data relating to the dissociation of monosubstituted benzoic acids in various solvents were extracted from the literature. The set of substituents included 25 common substituents in the meta position and the same number of substituents in the para position. Hydrogen served as the reference standard. The sets were subjected to regression analysis using conventional empirical models. The Hammett model was found to be valid within the limits of experimental error. The Taft model with the σR0, σR+ and σR- parameters is the best model to account for the substituent effects from the meta or para position solely. The inductive and mesomeric effects of the substituent are also best separated on this parametric scale. By applying the method of conjugated deviations (analysis of latent variables), a single latent variable was found to be sufficient to describe the data variability in all the three data sets analyzed (meta + para in the Hammett model and sets of meta and para substituted derivatives separately). The relationship between the first latent variables from the meta and para positions is isoparametric, the substituents lie on three straight lines intersecting in one point. The first straight line corresponds to substituents with the I effect (CH3, C2H5, tert-C4H9, C6H5, SO2NH2, CN, NO2 and hydrogen as the reference standard), the second straight line corresponds to substituents with I and +M effects (NH2, N(CH3)2, NHCOCH3, CH3O, SH, F, Cl, Br), and the third straight line corresponds to substituents with I and -M effects (CHO, CH3CO, COOR, SO2CH3, CF3). The +M mesomeric effect is twice as strong as the -M effect. These facts were used to propose a new empirical model for the description of substituent effects by means of one substituent constant and one (meta) or two (para) reaction constants. The PLS method revealed that the additional effects contribute about 8% to the data variability in the interpretation of the para substitution through meta substitution.
Methods to Reduce Concentration Polarization and Fouling in Membrane Filtration
Authors: Mikulášek Petr
Published: July 2002

Various methods and concepts that are currently being used and proposed to control or minimize concentration polarization and fouling in membrane separation processes are reviewed. A morphological analysis of hydrodynamic ways to prevent the detrimental influence on fluxes is given. The potentials of these different approaches are analyzed and some examples of module designs resulting from the various approaches with special attention to rotary membrane modules are given.
Phenanthridinium-Based Azomethine Ylides in 1,3-Dipolar Cycloaddition with Dimethyl Acetylenedicarboxylate
Authors: Potáček Milan , Topinka Tomáš , Dostál Jiří , Marek Jaromír
Published: July 2002

Azomethine ylides generated from 5-(alkoxycarbonylmethyl) phenanthridinium cations I were used in the 1,3-dipolar cycloadditions with dimethyl acetylenedicarboxylate as dipolarophile. The cycloadducts II were found to be very unstable species and readily oxidized to fully aromatic pyrrolo[1,2-f]phenanthridines III. Their structure was compared with the compounds formed by the oxidation of cycloadducts IV of the same ylide with dimethyl fumarate and maleate. In both cases pyrrolines V were found as intermediates. The structure of the products was determined by NMR and MS as well as by X-ray analysis.
Hugershoff Reaction of N-1- or N-2-Naphthoyl-N'-monosubstituted and N',N'-Disubstituted Thiourea Derivatives
Authors: Dzurilla Milan , Kutschy Peter , Imrich Ján , Brtoš Stanislav
Published: July 2002

Hugershoff reaction of N-1- or N-2-naphthoyl-N'-monosubstituted and N',N'-disubstituted thiourea derivatives, prepared by the reaction of 1-naphthoyl or 2-naphthoyl isothiocyanate with primary or secondary amines, was studied. Depending on substituents the corresponding urea or benzothiazole derivatives were obtained as reaction products. Their structure was determined on the basis of infrared, 1H and 13C NMR and mass spectra.