bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phosphorylation of p53: a Novel Pathway for p53 Inactivation in Human T-Cell Lymphotropic Virus Type 1-Transformed Cells
Authors: Pise-Masison Cynthia A. , Radonovich Michael , Sakaguchi Kazuyasu , Appella Ettore , Brady John N. 
Published: December 2019

ABSTRACT
Inhibition of p53 function, through either mutation or interaction with viral or cellular transforming proteins, correlates strongly with the oncogenic potential. Only a small percentage of human T-cell lymphotropic virus type 1 (HTLV-1)-transformed cells carry p53 mutations, and mutated p53 genes have been found in only one-fourth of adult T-cell leukemia cases. In previous studies, we demonstrated that wild-type p53 is stabilized and transcriptionally inactive in HTLV-1-transformed cells. Further, the viral transcriptional activator Tax plays a role in both the stabilization and inactivation of p53 through a mechanism involving the first 52 amino acids of p53. Here we show for the first time that phosphorylation of p53 inactivates p53 by blocking its interaction with basal transcription factors. Using two-dimensional peptide mapping, we demonstrate that peptides corresponding to amino acids 1 to 19 and 387 to 393 are hyperphosphorylated in HTLV-1-transformed cells. Moreover, using antibodies specific for phosphorylated Ser15 and Ser392, we demonstrate increased phosphorylation of these amino acids. Since HTLV-1 p53 binds DNA in a sequence-specific manner but fails to interact with TFIID, we tested whether phosphorylation of the N terminus of p53 affected p53-TFIID interaction. Using biotinylated peptides, we show that phosphorylation of Ser15 alone inhibits p53-TFIID interaction. In contrast, phosphorylation at Ser15 and -37 restores TFIID binding and blocks MDM2 binding. Our studies provide evidence that HTLV-1 utilizes the posttranslational modification of p53 in vivo to inactivate function of the tumor suppressor protein.
Herpes Simplex Virus Type 1 Tegument Protein VP22 Induces the Stabilization and Hyperacetylation of Microtubules
Authors: Elliott Gillian , O’Hare Peter 
Published: December 2019

ABSTRACT
The role of the herpes simplex virus type 1 tegument protein VP22 during infection is as yet undefined. We have previously shown that VP22 has the unusual property of efficient intercellular transport, such that the protein spreads from single expressing cells into large numbers of surrounding cells. We also noted that in cells expressing VP22 by transient transfection, the protein localizes in a distinctive cytoplasmic filamentous pattern. Here we show that this pattern represents a colocalization between VP22 and cellular microtubules. Moreover, we show that VP22 reorganizes microtubules into thick bundles which are easily distinguishable from nonbundled microtubules. These bundles are highly resistant to microtubule-depolymerizing agents such as nocodazole and incubation at 4°C, suggesting that VP22 has the capacity to stabilize the microtubule network. In addition, we show that the microtubules contained in these bundles are modified by acetylation, a marker for microtubule stability. Analysis of infected cells by both immunofluorescence and measurement of microtubule acetylation further showed that colocalization between VP22 and microtubules, and induction of microtubule acetylation, also occurs during infection. Taken together, these results suggest that VP22 exhibits the properties of a classical microtubule-associated protein (MAP) during both transfection and infection. This is the first demonstration of a MAP encoded by an animal virus.
Human T-Cell Leukemia Virus Type 1 Reverse Transcriptase (RT) Originates from the
pro
and
pol
Open Reading Frames and Requires the Presence of RT-RNase H (RH) and RT-RH-Integrase Proteins for Its Activity
Authors: Trentin Bernadette , Rebeyrotte Nicole , Mamoun Robert Z. 
Published: December 2019

ABSTRACT

The first description of an active form of a recombinant human T-cell leukemia virus type 1 (HTLV-1) reverse transcriptase (RT) and subsequent predictions of its amino acid sequence and quaternary structure are reported here. By using amino acid alignment methods, the NH
2
and COOH termini of the RT, RNase H (RH), and integrase (IN) domains of the Pol polyprotein were determined. The HTLV-1 RT seems to be unique since its NH
2
terminus is probably encoded by the
pro
open reading frame (ORF) fused downstream, via a transframe peptide, to the polypeptide encoded by the
pol
ORF. The HTLV-1 Pol amino acid sequence was revealed to be highly similar to that of Rous sarcoma virus (RSV), particularly at the RT-RH hinge region. These two domains remain linked for RSV; this may also be the case for HTLV-1. In light of these results, RT, RT-RH, and RT-RH-IN genes were constructed and introduced into His-tagged protein expression vectors. The corresponding proteins were synthesized in vitro, and the DNA polymerase activities of different protein combinations were tested. Solely the RT-RH–RT-RH-IN combination was found to have a significant activity level. Velocity sedimentation analysis suggested that the HTLV-1 RT-RH and RT-RH-IN monomers are likely associated in an oligomeric structure, probably of the α
3
/β type.
In Vivo Effects of Mutations in Woodchuck Hepatitis Virus Enhancer II
Authors: Wei Yu , Tennant Bud , Ganem Don 
Published: December 2019

ABSTRACT

Woodchuck hepatitis virus (WHV) enhancer II (EnII) is located upstream of the major pregenomic RNA promoter and is thought to play an important role in the insertional activation of the N-
myc2
gene during WHV hepatocarcinogenesis. WHV EnII is recognized by at least three host transcription factors: HNF-1, HNF-4, and Oct-1. Here, the roles of these EnII-binding factors in viral transcription and replication have been further examined. In HepG2 cells transiently transfected with a chloramphenicol acetyltransferase (CAT) gene whose expression is dependent upon EnII, mutations in either the HNF-1 or the HNF-4 site strongly reduced CAT activity, while ablation of the Oct-1 site decreased CAT expression only twofold. Mutations in more than one site completely abolished reporter expression. These same mutations were also tested in an overlength WHV genome for their impact on viral replication and gene expression. In transfected HepG2 cells, lesions in the HNF-1 site inactivated pregenomic RNA expression and viral reverse transcription, with only minimal effects on the expression of other viral mRNAs. By contrast, Oct-1 site lesions had no effect on either viral RNA synthesis or DNA replication, and HNF-4 site lesions produced a modest reduction of pregenomic RNA but had no impact on viral DNA synthesis. Testing of the mutants in susceptible woodchucks revealed that, as expected, viruses with lesions in the HNF-1 site were nearly noninfectious, while mutants with lesions at the Oct-1 site were fully replication competent. HNF-4 site mutants were replication competent but may display reduced levels of replication in the intact animal host. We conclude that (i) EnII is primarily devoted to the regulation of pregenomic RNA in WHV, (ii) HNF-1 is essential for EnII function in vivo, and (iii) HNF-4 plays a demonstrable but adjunctive role in EnII function.
Gastrointestinal T Lymphocytes Retain High Potential for Cytokine Responses but Have Severe CD4+T-Cell Depletion at All Stages of Simian Immunodeficiency Virus Infection Compared to Peripheral Lymphocytes
Authors: Smit-McBride Zeljka , Mattapallil Joseph J. , McChesney Michael , Ferrick David , Dandekar Satya 
Published: December 2019

ABSTRACTGastrointestinal complications in human immunodeficiency virus (HIV) infection are indicative of impaired intestinal mucosal immune system. We used simian immunodeficiency virus (SIV)-infected rhesus macaques as an animal model for HIV to determine pathogenic effects of SIV on intestinal T lymphocytes. Intestinal CD4+T-cell depletion and the potential for cytokine responses were examined during SIV infection and compared with results for lymphocytes from lymph nodes and blood. Flow cytometric analysis demonstrated severe depletion of CD4+CD8−single-positive T cells and CD4+CD8+double-positive T cells in intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) during primary SIV infection which persisted through the entire course of SIV infection. In contrast, CD4+T-cell depletion was gradual in peripheral lymph nodes and blood. Flow cytometric analysis of intracellular gamma interferon (IFN-γ) and interleukin-4 (IL-4) production following short-term mitogenic activation revealed that LPL retained same or higher capacity for IFN-γ production in all stages of SIV infection compared to uninfected controls, whereas peripheral blood mononuclear cells displayed a gradual decline. The CD8+T cells were the major producers of IFN-γ. There was no detectable change in the frequency of IL-4-producing cells in both LPL and peripheral blood mononuclear cells. Thus, severe depletion of CD4+LPL and IEL in primary SIV infection accompanied by altered cytokine responses may reflect altered T-cell homeostasis in intestinal mucosa. This could be a mechanism of SIV-associated enteropathy and viral pathogenesis. Dynamic changes in intestinal T lymphocytes were not adequately represented in peripheral lymph nodes or blood.
Steady-State Plasma Membrane Expression of Human Cytomegalovirus gB Is Determined by the Phosphorylation State of Ser
900
Authors: Fish Kenneth N. , Soderberg-Naucler Cecilia , Nelson Jay A. 
Published: December 2019

ABSTRACT

Human cytomegalovirus (HCMV) infection of an astrocytoma cell line (U373) or human fibroblast (HF) cells results in a differential cell distribution of the major envelope glycoprotein gB (UL55). This 906-amino-acid type I glycoprotein contains an extracellular domain with a signal sequence, a transmembrane domain, and a 135-amino-acid cytoplasmic tail with a consensus casein kinase II (CKII) site located at Ser
900
. Since phosphorylation of proteins in the secretory pathway is an important determinant of intracellular trafficking, the state of gB phosphorylation in U373 and HF cells was examined. Analysis of cells expressing wild-type gB and gB with site-specific mutations indicated that the glycoprotein was equally phosphorylated at a single site, Ser
900
, in both U373 and HF cells. To assess the effect of charge on gB surface expression in U373 cells, Ser
900
was replaced with an aspartate (Asp) or alanine (Ala) residue to mimic the phosphorylated and nonphosphorylated states, respectively. Expression of the Asp but not the Ala gB mutation resulted in an increase in the steady-state expression of gB at the plasma membrane (PM) in U373 cells. In addition, treatment of U373 cells with the phosphatase inhibitor tautomycin resulted in the accumulation of gB at the PM. Interestingly, the addition of a charge at Ser
900
trapped gB in a low-level cycling pathway at the PM, preventing trafficking of the protein to the
trans
-Golgi network or other intracellular compartments. Therefore, these results suggest that a tautomycin-sensitive phosphatase regulates cell-specific PM retrieval of gB to intracellular compartments.
Actinomycin D Inhibits Human Immunodeficiency Virus Type 1 Minus-Strand Transfer in In Vitro and Endogenous Reverse Transcriptase Assays
Authors: Guo Jianhui , Wu Tiyun , Bess Julian , Henderson Louis E. , Levin Judith G. 
Published: December 2019

ABSTRACT

In this report we demonstrate that human immunodeficiency virus type 1 (HIV-1) minus-strand transfer, assayed in vitro and in endogenous reactions, is greatly inhibited by actinomycin D. Previously we showed that HIV-1 nucleocapsid (NC) protein (a nucleic acid chaperone catalyzing nucleic acid rearrangements which lead to more thermodynamically stable conformations) dramatically stimulates HIV-1 minus-strand transfer by preventing TAR-dependent self-priming from minus-strand strong-stop DNA [(−) SSDNA]. Despite this potent activity, the addition of NC to in vitro reactions with actinomycin D results in only a modest increase in the 50% inhibitory concentration (IC
50
) for the drug. PCR analysis of HIV-1 endogenous reactions indicates that minus-strand transfer is inhibited by the drug with an IC
50
similar to that observed when NC is present in the in vitro system. Taken together, these results demonstrate that NC cannot overcome the inhibitory effect of actinomycin D on minus-strand transfer. Other experiments reveal that at actinomycin D concentrations which severely curtail minus-strand transfer, neither the synthesis of (−) SSDNA nor RNase H degradation of donor RNA is affected; however, the annealing of (−) SSDNA to acceptor RNA is significantly reduced. Thus, inhibition of the annealing reaction is responsible for actinomycin D-mediated inhibition of strand transfer. Since NC (but not reverse transcriptase) is required for efficient annealing, we conclude that actinomycin D inhibits minus-strand transfer by blocking the nucleic acid chaperone activity of NC. Our findings also suggest that actinomycin D, already approved for treatment of certain tumors, might be useful in combination therapy for AIDS.
Multiple Enzymatic Activities Associated with Recombinant NS3 Protein of Hepatitis C Virus
Authors: Gallinari Paola , Brennan Debra , Nardi Chiara , Brunetti Mirko , Tomei Licia , Steinkühler Christian , De Francesco Raffaele 
Published: December 2019

ABSTRACTThe hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein inEscherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.
Sequence Requirements for Removal of tRNA by an Isolated Human Immunodeficiency Virus Type 1 RNase H Domain
Authors: Smith Christine M. , Leon Oscar , Smith Jeffrey S. , Roth Monica J. 
Published: December 2019

ABSTRACT

Retroviral reverse transcriptase-associated RNase H enzymes are responsible for degradation of viral RNA, including removal of the tRNA primer after plus-strand strong-stop synthesis and cleavage of the polypurine tract primer. These activities are required for the complex viral replication and result in generation of the long terminal repeats. The human immunodeficiency virus type 1 (HIV-1) RNase H domain has been expressed independently of the polymerase domain and possesses Mn
2+
-dependent activity with a hexahistidine tag. The isolated domain maintains the ability to specifically remove a tRNA primer mimic. In this study, the substrate determinants for recognition of the cognate tRNA
3
Lys
are defined. Model substrates were constructed which mimic the RNA-DNA hybrid obtained from plus-strand strong-stop synthesis. Deletion substrates containing only 12, 9, or 6 positions of the tRNA primer were capable of being cleaved by the isolated RNase H domain. Mismatch and bromodeoxyuridine mutagenesis analysis indicated that positions 2, 3, 4, and 6, when mutated, affected the specificity of RNase H activity. Substitution substrates indicated that positions 4 and 6 within the RNA primer were important for recognition and cleavage by the HIV-1 isolated RNase H domain. Moloney murine leukemia virus-HIV-1 hybrid substrates were constructed which demonstrated that changes to HIV-1 sequences at positions 4 and 6 were sufficient but not optimal for regaining cleavage by the isolated HIV-1 RNase H domain. Optimal site-specific cleavage between the terminal ribonucleotide A and ribonucleotide C requires additional sequences beyond the first six positions but less than nine.
Human Immunodeficiency Virus Type 1 Genome Activation Induced by Human T-Cell Leukemia Virus Type 1 Tax Protein Is through Cooperation of NF-κB and Tat
Authors: Cheng Hua , Tarnok Jennifer , Parks Wade P. 
Published: December 2019

ABSTRACTFor productive replication of human immunodeficiency virus type 1 (HIV-1) in host cells, the viral genome-encoded transactivator Tat and several cellular transcription factors are required for efficient viral gene transcription. However, it remains unclear how the viral genome initiates transcription before Tat is transcribed or when Tat is at suboptimal levels. Here, we utilized the human T-cell leukemia type 1 Tax protein as a molecular tool to investigate the mechanism of viral gene transcription that initiates the early phase of infection of HIV-1. Tax alone does not significantly increase the activity of HIV-1 long terminal repeat (LTR) in T lymphocytes, but it markedly enhanced the replication of an infectious HIV-1 provirus with a truncatednefgene. This enhancement is preferentially mediated by the cooperation of Tax and Tat which is dependent on TAR and duplicated κBciselements of the HIV-1 LTR as well as the NF-κB activation domain of Tax. Furthermore, phorbol myristate acetate and membrane-targeted HIV-1 Nef also enhanced the LTR activity in the presence of Tat in the TAR- and κBciselement-dependent manner. These data suggest that activated NF-κB can functionally interact with a suboptimal amount of Tat and the HIV-1 LTR for efficient initiation of viral gene transcription.
Viral Determinants of Rotavirus Pathogenicity in Pigs: Evidence that the Fourth Gene of a Porcine Rotavirus Confers Diarrhea in the Homologous Host
Authors: Bridger Janice C. , Tauscher Gabriele I. , Desselberger Ulrich 
Published: December 2019

ABSTRACT
A porcine rotavirus (prv) monoreassortant, S-F4, which carries RNA segment 4 of the pig-pathogenic variant prv 4F in the genetic background of the pig-apathogenic variant prv 4S (G. I. Tauscher and U. Desselberger, J. Virol. 71:853–857, 1997), was found to be pathogenic in gnotobiotic piglets. This indicates that RNA segment 4 of the pig-pathogenic variant prv 4F is a major determinant of pathogenicity in its homologous host.
The Second-Site Mutation in the Herpes Simplex Virus Recombinants Lacking the γ
1
34.5 Genes Precludes Shutoff of Protein Synthesis by Blocking the Phosphorylation of eIF-2α
Authors: Cassady Kevin A. , Gross Martin , Roizman Bernard 
Published: December 2019

ABSTRACT

In cells infected with the herpes simplex virus 1 (HSV-1) recombinant R3616 lacking both copies of the γ
1
34.5 gene, the double-stranded protein kinase R (PKR) is activated, eIF-2α is phosphorylated, and protein synthesis is shut off. Although PKR is also activated in cells infected with the wild-type virus, the product of the γ
1
34.5 gene, infected-cell protein 34.5 (ICP34.5), binds protein phosphatase 1α and redirects it to dephosphorylate eIF-2α, thus enabling sustained protein synthesis. Serial passage in human cells of a mutant lacking the γ
1
34.5 gene yields second-site, compensatory mutants lacking various domains of the α47 gene situated next to the U
S
11 gene (I. Mohr and Y. Gluzman, EMBO J. 15:4759–4766, 1996). We report the construction of two recombinant viruses: R5103, lacking the γ
1
34.5, U
S
8, -9, -10, and -11, and α47 (U
S
12) genes; and R5104, derived from R5103 and carrying a chimeric DNA fragment containing the U
S
10 gene and the promoter of the α47 gene fused to the coding domain of the U
S
11 gene. R5104 exhibited a protein synthesis profile similar to that of wild-type virus, whereas protein synthesis was shut off in cells infected with R5103 virus. Studies on the wild-type parent and mutant viruses showed the following: (i) PKR was activated in cells infected with parent or mutant virus but not in mock-infected cells, consistent with earlier studies; (ii) lysates of R3616, R5103, and R5104 virus-infected cells lacked the phosphatase activity specific for eIF-2α characteristic of wild-type virus-infected cells; and (iii) lysates of R3616 and R5103, which lacked the second-site compensatory mutation, contained an activity which phosphorylated eIF-2α in vitro, whereas lysates of mock-infected cells or cells infected with HSV-1(F) or R5104 did not phosphorylate eIF-2α. We conclude that in contrast to wild-type virus-infected cells, which preclude the shutoff of protein synthesis by causing rapid dephosphorylation of eIF-2α, in cells infected with γ
1
34.5
−
virus carrying the compensatory mutation, eIF-2α is not phosphorylated. The activity made apparent by the second-site mutation may represent a more ancient mechanism evolved to preclude the shutoff of protein synthesis.
The Vaccinia Virus A18R DNA Helicase Is a Postreplicative Negative Transcription Elongation Factor
Authors: Xiang Ying , Simpson David A. , Spiegel Jason , Zhou Aimin , Silverman Robert H. , Condit Richard C. 
Published: December 2019

ABSTRACT
Loss of vaccinia virus A18R gene function results in an aberrant transcription profile termed promiscuous transcription, defined as transcription within regions of the genome which are normally transcriptionally silent late during infection. Promiscuous transcription results in an increase in the intracellular concentration of double-stranded RNA, which in turn results in activation of the cellular 2-5A pathway and subsequent RNase L-catalyzed degradation of viral and cellular RNAs. One of three hypotheses could account for promiscuous transcription: (i) reactivation of early promoters late during infection, (ii) random transcription initiation, (iii) readthrough transcription from upstream promoters. Transcriptional analysis of several viral genes, presented here, argues strongly against the first two hypotheses. We have tested the readthrough hypothesis by conducting a detailed transcriptional analysis of a region of the vaccinia virus genome which contains three early genes (M1L, M2L, and K1L) positioned directly downstream of the intermediate gene, K2L. The results show that mutation of the A18R gene results in increased readthrough transcription of the M1L gene originating from the K2L intermediate promoter. A18R mutant infection of RNase L knockout mouse fibroblast (KO3) cells does not result in 2-5A pathway activation, yet the virus mutant is defective in late viral gene expression and remains temperature sensitive. These results demonstrate that the A18R gene product is a negative transcription elongation factor for postreplicative viral genes.