bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of Moloney Murine Leukemia Virus RNase H Rescues the Growth Defect of an
Escherichia coli
Mutant
Authors: Campbell Andrew G. 
Published: July 2002

ABSTRACT

A 157-amino-acid fragment of Moloney murine leukemia virus reverse transcriptase encoding RNase H is shown to rescue the growth-defective phenotype of an
Escherichia coli
mutant. In vitro assays of the recombinant wild-type protein purified from the conditionally defective mutant confirm that it is catalytically active. Mutagenesis of one of the presumptive RNase H-catalytic residues results in production of a protein variant incapable of rescue and which lacks activity in vitro. Analyses of additional active site mutants demonstrate that their encoded variant proteins lack robust activity yet are able to rescue the bacterial mutant. These results suggest that genetic complementation may be useful for in vivo screening of mutant viral RNase H gene fragments and in evaluating their function under conditions that more closely mimic physiological conditions. The rescue system may also be useful in verifying the functional outcomes of mutations based on protein structural predictions and modeling.
MultiplecisRegulatory Elements Control RANTES Promoter Activity in Alveolar Epithelial Cells Infected with Respiratory Syncytial Virus
Authors: Casola Antonella , Garofalo Roberto P. , Haeberle Helene , Elliott Todd F. , Lin Rongtuan , Jamaluddin Mohammad , Brasier Allan R. 
Published: July 2002

ABSTRACTRespiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part through its ability to induce chemokine synthesis in infected airway epithelial cells. RANTES (regulated upon activation, normally T-cell expressed and presumably secreted) is a CC chemokine which recruits and activates monocytes, lymphocytes, and eosinophils, all cell types present in the lung inflammatory infiltrate induced by RSV infection. In this study, we analyzed the mechanism of RSV-induced RANTES promoter activation in human type II alveolar epithelial cells (A549 cells). Promoter deletion and mutagenesis experiments indicate that RSV requires the presence of five differentcisregulatory elements, located in the promoter fragment spanning from −220 to +55 nucleotides, corresponding to NF-κB, C/EBP, Jun/CREB/ATF, and interferon regulatory factor (IRF) binding sites. Although site mutations of the NF-κB, C/EBP, and CREB/AP-1 like sites reduce RSV-induced RANTES gene transcription to 50% or less, only mutations affecting IRF binding completely abolish RANTES inducibility. Supershift and microaffinity isolation assays were used to identify the different transcription factor family members whose DNA binding activity was RSV inducible. Expression of dominant negative mutants of these transcription factors further established their central role in virus-induced RANTES promoter activation. Our finding that the presence of multiplecisregulatory elements is required for full activation of the RANTES promoter in RSV-infected alveolar epithelial cells supports the enhanceosome model for RANTES gene transcription, which is absolutely dependent on binding of IRF transcription factors. The identification of regulatory mechanisms of RANTES gene expression is fundamental for rational design of inhibitors of RSV-induced lung inflammation.
Mutations That Increase In Situ Priming Also Decrease Circularization for Duck Hepatitis B Virus
Authors: Loeb Daniel D. , Tian Ru 
Published: July 2002

ABSTRACT
The process of hepadnavirus reverse transcription involves two template switches during the synthesis of plus-strand DNA. The first involves translocation of the plus-strand primer from its site of generation, the 3′ end of minus-strand DNA, to the complementary sequence DR2, located near the 5′ end of the minus-strand DNA. Plus strands initiated from DR2 are extended to the 5′ end of the minus-strand DNA. At this point, the 3′ end of the minus strand becomes the template via the second template switch, a process called circularization. Elongation of circularized plus-strand DNA generates relaxed circular DNA. Although most virions contain relaxed circular DNA, some contain duplex linear DNA. Duplex linear genomes are synthesized when the plus-strand primer is used at the site of its generation, the 3′ end of the minus-strand template. This type of synthesis is called in situ priming. Although in situ priming is normally low, in some duck hepatitis B virus mutants this type of priming is elevated. For example, mutations within the 3′ end of the minus-strand DNA can lead to increased levels of in situ priming. We report here that these same mutations result in a second defect, a less efficient template switch that circularizes the genome. Although it is not clear how these mutations affect both steps in DNA replication, our findings suggest a commonality in the mechanism of initiation of plus-strand synthesis and the template switch that circularizes the genome.
Destabilization of the Retinoblastoma Tumor Suppressor by Human Papillomavirus Type 16 E7 Is Not Sufficient To Overcome Cell Cycle Arrest in Human Keratinocytes
Authors: Helt Anna-Marija , Galloway Denise A. 
Published: July 2002

ABSTRACT
The E7 oncoprotein of human papillomavirus type 16 promotes cell proliferation in the presence of antiproliferative signals. Mutagenesis of E7 has revealed that this activity requires three regions, conserved regions 1 and 2 and a C-terminal zinc finger. Binding to the retinoblastoma tumor repressor (Rb) through an LxCxE motif in conserved region 2 is necessary, but not sufficient, for E7 to induce proliferation. We tested the hypothesis that binding to Rb is not sufficient because conserved region 1 and/or the C terminus are required for E7 to functionally inactivate Rb and thus induce proliferation. One mechanism proposed for how E7 inactivates Rb is by blocking Rb-E2F binding. Either conserved region 1 or the C terminus was necessary, in combination with the LxCxE motif, for E7 to block Rb-E2F binding in vitro. While all full-length E7 proteins with mutations outside of the LxCxE motif inhibited Rb-E2F binding, some failed to abrogate cell cycle arrest, demonstrating that blocking Rb-E2F binding is not sufficient for abrogating antiproliferative signals. Another mechanism proposed for how E7 inactivates Rb is by promoting the destabilization of Rb protein. Mutations in conserved region 1 or the LxCxE motif prevented E7 from reducing the half-life of Rb. Though no specific C-terminal residues of E7 were essential for destabilizing Rb, a novel class of mutations that uncouple the destabilization of Rb from the deregulation of keratinocyte proliferation was discovered. Destabilization of Rb correlated with the abrogation of Rb-induced quiescence but was not sufficient for overriding DNA damage-induced cell cycle arrest or for increasing keratinocyte life span. Finally, the same regions of E7 required for destabilizing Rb were required for reducing p107 and p130 levels. Together, these results suggest that inactivation of all three Rb family members is not sufficient to deregulate keratinocyte cell cycle control.
Therapeutic Effect of a Gag-Nuclease Fusion Protein against Retroviral Infection In Vivo
Authors: Schumann Gerald , Hermankova Monika , Cannon Keith , Mankowski Joseph L. , Boeke Jef D. 
Published: July 2002

ABSTRACTRecently, remarkable progress has been made in developing effective combination drug therapies that can control but not cure retroviral replication. Even when effective, these drug regimens are toxic, they require demanding administration schedules, and resistant viruses can emerge. Thus the need for new gene-based therapies continues. In one such approach, capsid-targeted viral inactivation (CTVI), nucleases fused to viral coat proteins are expressed in infected cells and become incorporated during virion assembly. CTVI can eliminate infectious murine retrovirus titer in tissue culture. Here we describe transgenic mice expressing fusions of the Moloney murine leukemia virus (Mo-MuLV) Gag protein to staphylococcal nuclease. This work tests the protective effect and demonstrates in vivo proof-of-principle of CTVI in transgenic mice expressing endogenous proviral copies of Mo-MuLV. The antiviral protein-expressing mice are phenotypically normal, attesting to the lack of toxicity of the fusion protein. The Mo-MuLV infection was much less virulent in transgenic littermates than in nontransgenic littermates. Gag-nuclease expression reduced infectious titers in blood up to 10-fold, decreased splenomegaly and leukemic infiltration, and increased life spans up to 2.5-fold in transgenic relative to nontransgenic infected animals. These results suggest that gene therapies based on similar fusion proteins, designed to attack human immunodeficiency virus or other retroviruses, could provide substantial therapeutic benefits.
Characterization of New Syncytium-Inhibiting Monoclonal Antibodies Implicates Lipid Rafts in Human T-Cell Leukemia Virus Type 1 Syncytium Formation
Authors: Niyogi Kakoli , Hildreth James E. K. 
Published: July 2002

ABSTRACTWe have previously shown that erythroleukemia cells (K562) transfected with vascular adhesion molecule 1 (VCAM-1) are susceptible to human T-cell leukemia virus type 1 (HTLV-1)-induced syncytium formation. Since expression of VCAM-1 alone is not sufficient to render cells susceptible to HTLV-1 fusion, K562 cells appear to express a second molecule critical for HTLV-induced syncytium formation. By immunizing mice with K562 cells, we have isolated four monoclonal antibodies (MAbs), K5.M1, K5.M2, K5.M3, and K5.M4, that inhibit HTLV-induced syncytium formation between infected MT2 cells and susceptible K562/VCAM1 cells. These MAbs recognize distinct proteins on the surface of cells as determined by cell phenotyping, immunoprecipitation, and Western blot analysis. Since three of the proteins recognized by the MAbs appear to be GPI linked, we isolated lipid rafts and determined by immunoblot analysis that all four MAbs recognize proteins that sort entirely or in large part to lipid rafts. Dispersion of lipid rafts on the cells by cholesterol depletion with β-cyclodextrin resulted in inhibition of syncytium formation, and this effect was not seen when the β-cyclodextrin was preloaded with cholesterol before treating the cells. The results of these studies suggest that lipid rafts may play an important role in HTLV-1 syncytium formation.
The Envelope Glycoprotein of Friend Spleen Focus-Forming Virus Covalently Interacts with and Constitutively Activates a Truncated Form of the Receptor Tyrosine Kinase Stk
Authors: Nishigaki Kazuo , Thompson Delores , Hanson Charlotte , Yugawa Takashi , Ruscetti Sandra 
Published: July 2002

ABSTRACT

The Friend spleen focus-forming virus (SFFV) encodes a unique envelope glycoprotein, gp55, which allows erythroid cells to proliferate and differentiate in the absence of erythropoietin (Epo). SFFV gp55 has been shown to interact with the Epo receptor complex, causing constitutive activation of various signal-transducing molecules. When injected into adult mice, SFFV induces a rapid erythroleukemia, with susceptibility being determined by the host gene
Fv-2
, which was recently shown to be identical to the gene encoding the receptor tyrosine kinase Stk/Ron. Susceptible, but not resistant, mice encode not only full-length Stk but also a truncated form of the kinase, sf-Stk, which may mediate the biological effects of SFFV infection. To determine whether expression of SFFV gp55 leads to the activation of sf-Stk, we expressed sf-Stk, with or without SFFV gp55, in hematopoietic cells expressing the Epo receptor. Our data indicate that sf-Stk interacts with SFFV gp55 as well as gp55
P
, the biologically active form of the viral glycoprotein, forming disulfide-linked complexes. This covalent interaction, as well as noncovalent interactions with SFFV gp55, results in constitutive tyrosine phosphorylation of sf-Stk and its association with multiple tyrosine-phosphorylated signal-transducing molecules. In contrast, neither Epo stimulation in the absence of SFFV gp55 expression nor expression of a mutant of SFFV that cannot interact with sf-Stk was able to induce tyrosine phosphorylation of sf-Stk or its association with any signal-transducing molecules. Covalent interaction of sf-Stk with SFFV gp55 and constitutive tyrosine phosphorylation of sf-Stk can also be detected in an erythroleukemia cell line derived from an SFFV-infected mouse. Our results suggest that SFFV gp55 may mediate its biological effects in vivo by interacting with and activating a truncated form of the receptor tyrosine kinase Stk.
Effect of Alpha Interferon on the Hepatitis C Virus Replicon
Authors: Guo Ju-Tao , Bichko Vadim V. , Seeger Christoph 
Published: July 2002

ABSTRACT
Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-α) and ribavirin combination therapy. The mechanism of the IFN-α response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-α reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-α independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-α. Finally, under our conditions, selection for IFN-α-resistant variants did not occur.
Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro
Authors: Kautz Armin R. , Weisshart Klaus , Schneider Annerose , Grosse Frank , Nasheuer Heinz-Peter 
Published: July 2002

ABSTRACT
Although p48 is the most conserved subunit of mammalian DNA polymerase α-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.
Recombinant Herpes Simplex Virus Type 1 Expressing Murine Interleukin-4 Is Less Virulent than Wild-Type Virus in Mice
Authors: Ghiasi Homayon , Osorio Yanira , Perng Guey-Chuen , Nesburn Anthony B. , Wechsler Steven L. 
Published: July 2002

ABSTRACTThe effect of interleukin-4 (IL-4) on herpes simplex virus type 1 (HSV-1) infection in mice was evaluated by construction of a recombinant HSV-1 expressing the gene for murine IL-4 in place of the latency-associated transcript (LAT). The mutant virus (HSV-IL-4) expressed high levels of IL-4 in cultured cells. The replication of HSV-IL-4 in tissue culture and in trigeminal ganglia was similar to that of wild-type virus. In contrast, HSV-IL-4 appeared to replicate less well in mouse eyes and brains. Although BALB/c mice are highly susceptible to HSV-1 infection, ocular infection with HSV-IL-4 resulted in 100% survival. Furthermore, 57% of the mice survived coinfection with a mixture of HSV-IL-4 and a lethal dose of wild-type McKrae, compared with only 10% survival following infection with McKrae alone. Similar to wild-type BALB/c mice, 100% of IL-4−/−mice also survived HSV-IL-4 infection. T-cell depletion studies suggested that protection against HSV-IL-4 infection was mediated by a CD4+-T-cell response.
Additive Effects Characterize the Interaction of Antibodies Involved in Neutralization of the Primary Dualtropic Human Immunodeficiency Virus Type 1 Isolate 89.6
Authors: Verrier Florence , Nádas Arthur , Gorny Miroslaw K. , Zolla-Pazner Susan 
Published: July 2002

ABSTRACT

Human immunodeficiency virus-type I (HIV-1) infection elicits antibodies (Abs) directed against several regions of the gp120 and gp41 envelope glycoproteins. Many of these Abs are able to neutralize T-cell-line-adapted strains (TCLA) of HIV-1, but only a few effectively neutralize primary HIV-1 isolates. The nature of HIV-1 neutralization has been carefully studied using human monoclonal Abs (MAbs), and the ability of such MAbs to act in synergy to neutralize HIV-1 has also been extensively studied. However, most synergy studies have been conducted using TCLA strains. To determine the nature of Ab interaction in HIV-1 primary isolate neutralization, a panel of 12 anti-HIV-1 human immunoglobulin G (IgG) MAbs, specific for epitopes in gp120 and gp41, were used. Initial tests showed that six of these MAbs, as well as sCD4, used individually, were able to neutralize the dualtropic primary isolate HIV-1
89.6
; MAbs giving significant neutralization at 2 to 10 μg/ml included 2F5 (anti-gp41), 50-69 (anti-gp41), IgG1b12 (anti-gp120
CD4bd
), 447-52D (anti-gp120
V3
), 2G12 (anti-gp120), and 670-D (anti-gp120
C5
). For studies of reagent interaction, 16 binary combinations of reagents were tested for their ability to neutralize HIV-1
89.6
. Reagent combinations tested included one neutralizing MAb with sCD4, six pairs consisting of two neutralizing MAbs, and nine pairs consisting of one neutralizing MAb with another non-neutralizing MAb. To assess the interaction of the latter type of combination, a new mathematical treatment of reagent interaction was developed since previously used methods could be used only when both reagents neutralize. Synergy was noted between sCD4 and a neutralizing anti-gp120
V3
MAb. Antagonism was noted between two pairs of anti-gp41 MAbs (one neutralizing and one non-neutralizing). All of the other 13 pairs of MAbs tested displayed only additive effects. These studies suggest that Abs rarely act in synergy to neutralize primary isolate HIV-1
89.6
; many anti-HIV-1 Abs act additively to mediate this biological function.
Characterization of a Porcine Lung Epithelial Cell Line Suitable for Influenza Virus Studies
Authors: Seo Sang Heui , Goloubeva Olga , Webby Richard , Webster Robert G. 
Published: July 2002

ABSTRACTWe established a porcine lung epithelial cell line designated St. Jude porcine lung cells (SJPL) and demonstrated that all tested influenza A and B viruses replicated in this cell line. The infectivity titers of most viruses in SJPL cells were comparable to or better than those in MDCK cells. The propagation of influenza viruses from clinical samples in SJPL cells did not lead to antigenic changes in the hemagglutinin molecule. The numbers of both Sia2-3Gal and Sia2-6Gal receptors on SJPL cells were greater than those on MDCK cells. Influenza virus infection of SJPL cells did not lead to apoptosis, as did infection of MDCK cells. No porcine endogenous retrovirus was detected in SJPL cells, and in contrast to MDCK cells, SJPL cells did not cause tumors in nude mice.
Infection of Nondividing Cells by Rous Sarcoma Virus
Authors: Hatziioannou Theodora , Goff Stephen P. 
Published: July 2002

ABSTRACT
A direct comparison demonstrates that Rous sarcoma virus is capable of infecting aphidicolin-arrested cells 10-fold more efficiently than murine leukemia virus but less efficiently than human immunodeficiency virus. The efficiency of infection of nondividing cells by the three viruses correlates with the respective ability of each viral DNA to enter the nucleus.
Two-Step Nature of Human T-Cell Leukemia Virus Type 1 Replication in Experimentally Infected Squirrel Monkeys (
Saimiri sciureus
)
Authors: Mortreux Franck , Kazanji Mirdad , Gabet Anne-Sophie , de Thoisy Benoit , Wattel Eric 
Published: July 2002

ABSTRACT

After experimental infection of squirrel monkeys (
Saimiri sciureus
) with human T-cell leukemia virus type 1 (HTLV-1)-infected cells, the virus is transcribed only transiently in circulating blood, spleen, and lymph nodes. Stable disappearance of viral expression occurs at 2 to 3 weeks after inoculation. This coincides with the development of the anti-HTLV-1 immune response and persistent detection of the provirus in peripheral blood mononuclear cells (PBMCs). In this study, the HTLV-1 replication pattern was analyzed over time in PBMCs and various organs from two HTLV-1-infected squirrel monkeys. Real-time quantitative PCR confirmed that PBMCs and lymphoid organs constitute the major reservoirs for HTLV-1. The PCR amplification of HTLV-1 flanking sequences from PBMCs evidenced a pattern of clonal expansion of infected cells identical to that observed in humans. Dissemination of the virus in body compartments appeared to result from cellular transport of the integrated provirus. The circulating proviral burden increased as a function of time in one animal studied over a period of 4 years. The high proviral loads observed in the last samples resulted from the accumulation of infected cells via the extensive proliferation of a restricted number of persistent clones on a background of polyclonally expanded HTLV-1-positive cells. Therefore, HTLV-1 primary infection in squirrel monkeys is a two-step process involving a transient phase of reverse transcription followed by persistent multiplication of infected cells. This suggests that the choice of the target for blocking HTLV-1 replication might depend on the stage of infection.