bluuebunny/crossref_metadata_2025_split huggingface dataset:
Viral Immediate-Early Proteins Abrogate the Modification by SUMO-1 of PML and Sp100 Proteins, Correlating with Nuclear Body Disruption
Authors: Müller Stefan , Dejean Anne 
Published: December 2019

ABSTRACTPML nuclear bodies (NBs) are subnuclear structures whose integrity is compromised in certain human diseases, including leukemia and neurodegenerative disorders. Infection by a number of DNA viruses similarly triggers the reorganization of these structures, suggesting an important role for the NBs in the viral infection process. While expression of the adenovirus E4 ORF3 protein leads to only a moderate redistribution of PML to filamentous structures, the herpes simplex virus (HSV) ICP0 protein and the cytomegalovirus (CMV) IE1 protein both induce a complete disruption of the NB structure. Recently, we and others have shown that the NB proteins PML and Sp100 are posttranslationally modified by covalent linkage with the ubiquitin-related SUMO-1 protein and that this modification may promote the assembly of these structures. Here we show that the HSV ICP0 and CMV IE1 proteins specifically abrogate the SUMO-1 modification of PML and Sp100, whereas the adenovirus E4 ORF3 protein does not affect this process. The potential of ICP0 and IE1 to alter SUMO-1 modification is directly linked to their capacity to disassemble NBs, thus strengthening the role for SUMO-1 conjugation in maintenance of the structural integrity of the NBs. This observation supports a model in which ICP0 and IE1 disrupt the NBs either by preventing the formation or by degrading of the SUMO-1-modified PML and Sp100 protein species. Finally, we show that the IE1 protein itself is a substrate for SUMO-1 modification, thus representing the first viral protein found to undergo this new type of posttranslational modification.
A Single Amino Acid Substitution in the Phosphoprotein of Respiratory Syncytial Virus Confers Thermosensitivity in a Reconstituted RNA Polymerase System
Authors: Marriott Anthony C. , Wilson Steven D. , Randhawa Jaspal S. , Easton Andrew J. 
Published: December 2019

ABSTRACT

The single amino acid change Gly172 to Ser in the phosphoprotein (P) of respiratory syncytial virus (RSV) has previously been shown to be responsible for the thermosensitivity and protein-negative phenotype of
ts
N19, a mutant of the B subgroup RSN-2 strain. This single change was inserted into the P gene of the A subgroup virus RSS-2, and the resulting phenotype was observed in a plasmid-driven reconstituted RSV RNA polymerase system. Expression from a genome analogue containing two reporter genes was thermosensitive when directed by plasmids containing the N, L, M2, and mutant P genes cloned under the control of T7 promoters. Analysis of RNA synthesis showed that mutant P protein was unable to produce genome, antigenome, or mRNA at the restrictive temperature. At a semipermissive temperature, genome, antigenome, and mRNA synthesis were all reduced, 6- to 30-fold, relative to synthesis directed by a wild-type P plasmid. Binding of the mutant P protein to N protein in the absence of other viral proteins was unaffected by temperature, indicating that the lesion did not produce a large enough structural change to disrupt this binding. These data suggest that the plasmid rescue system is suitable for investigation of the role of thermosensitive mutations in RSV polymerase components in RNA synthesis.
Human Immunodeficiency Virus Type 2 Produces a Defect in CD3-γ Gene Transcripts Similar to That Observed for Human Immunodeficiency Virus Type 1
Authors: Segura Iris , Delmelle-Wibaut Christine , Janssens Michel , Cleuter Yvette , van den Broeke Anne , Kettmann Richard , Willard-Gallo Karen E. 
Published: December 2019

ABSTRACTT cells are central players in the immune response to infectious disease, with the specificity of their responses controlled by the T-cell receptor (TCR)/CD3 complex on the cell surface. Impairment of TCR/CD3-directed CD4+T-cell immune responses is frequently observed in individuals infected with human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). Virus replication is also regulated by T-cell activation factors, with HIV-1 and HIV-2 responding to different TCR/CD3-directed cellular pathways. We previously demonstrated that HIV-1 infection of the human interleukin-2-dependent CD4+T-cell line WE17/10 abrogates TCR/CD3 function and surface expression by a specific loss of CD3-γ gene transcripts. In this study, we show that HIV-2 provokes the same molecular defect in CD3-γ gene transcripts, resulting in a similar but delayed progressive loss of TCR/CD3 surface expression after infection.
Epstein-Barr Virus (EBV) Nuclear Protein 2-Induced Disruption of EBV Latency in the Burkitt’s Lymphoma Cell Line Akata: Analysis by Tetracycline-Regulated Expression
Authors: Fujiwara Shigeyoshi , Nitadori Yoshikazu , Nakamura Hiroyuki , Nagaishi Takashi , Ono Yasushi 
Published: December 2019

ABSTRACTThe Burkitt’s lymphoma (BL) cell line Akata retains the latency I program of Epstein-Barr virus (EBV) gene expression and cross-linking of its surface immunoglobulin G (IgG) by antibodies results in activation of viral replication. When EBV nuclear antigen 2 (EBNA2) was artificially expressed by a constitutive expression vector, the Cp EBNA promoter remained inactive and accordingly the latency III program was not induced. In contrast, expression of LMP2A and activity of the Fp lytic promoter were activated. Consistent with this Fp activity, the rate of spontaneous activation of the EBV replicative cycle was increased significantly, suggesting the possibility that EBNA2 can induce EBV replication. The efficiency of anti-IgG-induced activation of the viral replication was reduced in Akata cells expressing EBNA2. To obtain more direct evidence for EBNA2-induced activation of the EBV replicative cycle, this protein was next expressed by a tetracycline-regulated expression system. EBNA2 was undetectable with low doses (<0.5 μg/ml) of tetracycline, while its expression was rapidly induced after removal of the antibiotic. This induced expression of EBNA2 was immediately followed by expression of EBV replicative cycle proteins in up to 50% of the cells, as shown by indirect immunofluorescence and immunoblot analysis. These results suggest an unexpected potential of EBNA2 to disrupt EBV latency and to activate viral replication.
Infection of Chicken Embryonic Fibroblasts by Measles Virus: Adaptation at the Virus Entry Level
Authors: Escoffier Carine , Gerlier Denis 
Published: December 2019

ABSTRACT
Measles virus (MV) has a tropism restricted to humans and primates and uses the human CD46 molecule as a cellular receptor. MV has been adapted to grow in chicken embryonic fibroblasts (CEF) and gave rise to an attenuated live vaccine. Hallé and Schwarz MV strains were compared in their ability to infect both simian Vero cells and CEF. Whereas both strains infected Vero cells, only the CEF-adapted Schwarz strain was able to efficiently infect CEF. Since the expression of the human MV receptor CD46 rendered the chicken embryonic cell line TCF more permissive to the infection by the Hallé MV strain, the MV entry into CEF appeared to be a limiting step in the absence of prior MV adaptation. CEF lacked reactivity with anti-CD46 antibodies but were found to express another protein allowing MV binding as an alternative receptor to CD46.
Primary Virus Envelope Cross-Reactivity of the Broadening Neutralizing Antibody Response during Early Chronic Human Immunodeficiency Virus Type 1 Infection
Authors: Zhang Peng Fei , Chen Xi , Fu Da Wei , Margolick Joseph B. , Quinnan Gerald V. 
Published: December 2019

ABSTRACTTo test the hypothesis that changing neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1) during chronic infection were a response to emergence of neutralization escape mutants, we cloned expressed and characterized envelope clones from patients in the Multicenter AIDS Cohort Study (MACS). Pseudotyped HIV-1 envelope clones obtained from differing time points were assessed for sensitivity to neutralization by using sera from different times from the same and different patients. Clones from early and late time points during chronic infection had similar neutralization sensitivity, and neutralizing antibody responses cross-reacted with early, late, and heterologous envelopes. The potential for broadly effective HIV-1 immunization is supported.
An Orphan G Protein-Coupled Receptor, GPR1, Acts as a Coreceptor To Allow Replication of Human Immunodeficiency Virus Types 1 and 2 in Brain-Derived Cells
Authors: Shimizu Nobuaki , Soda Yasushi , Kanbe Katsuaki , Liu Hui-Yu , Jinno Atsushi , Kitamura Toshio , Hoshino Hiroo 
Published: December 2019

ABSTRACTTwelve G protein-coupled receptors, including chemokine receptors, act as coreceptors and determinants for the cell tropisms of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). We isolated HIV-1 variants from T-cell-line (T)- and macrophage (M)-tropic (i.e., dualtropic) (R5-R3-X4) HIV-1 strains and also produced six HIV-1 mutants carrying single-point amino acid substitutions at the tip of the V3 region of the Env protein of HIV-1. These variants and three mutants infected brain-derived CD4-positive cells that are resistant to M-, T-, or dualtropic (R5, X4, or R5-X4) HIV-1 strains. However, a factor that determines this cell tropism has not been identified. This study shows that primary brain-derived fibroblast-like cell strains, BT-3 and BT-20/N, as well as a CD4-transduced glioma cell line, U87/CD4, which were susceptible to these HIV-1 variants and mutants and the HIV-2RODstrain, expressed mRNA of an orphan G protein-coupled receptor (GPCR), GPR1. When a CD4-positive cell line which was strictly resistant to infection with diverse HIV-1 and HIV-2 strains was transduced with GPR1, the cell line became susceptible to these HIV-1 variants and mutants and to an HIV-2 strain but not to T- or dualtropic HIV-1 strains, and numerous syncytia formed after infection. These results indicate that GPR1 functions as a coreceptor for the HIV-1 variants and mutants and for the HIV-2RODstrain in vitro.
Variants from the Diverse Virus Population Identified at Seroconversion of a Clade A Human Immunodeficiency Virus Type 1-Infected Woman Have Distinct Biological Properties
Authors: Poss Mary , Overbaugh Julie 
Published: December 2019

ABSTRACTDevelopment of effective therapeutics to prevent new infections with human immunodeficiency type 1 (HIV-1) is predicated on an understanding of the properties that provide a selective advantage to a transmitted viral population. In contrast to the homogeneous virus population that typifies early HIV-1 infection of men, the viral population in women recently infected with clade A HIV-1 is genetically diverse, based on evaluation of the envelope gene. A longitudinal study of viral envelope evolution in several women suggested that representative envelope variants detected at seroconversion had distinct biological properties that affected viral fitness. To test this hypothesis, a full-length, infectious molecular clone, Q23-17, was obtained from an infected woman 1 year following seroconversion, and chimeric viruses containing envelope genes representative of seroconversion and 27-month-postseroconversion populations were constructed. Dendritic cells (DC) could transfer infection of seroconversion variant Q23ScA, which dominated the viral population in the year following seroconversion, and the closely related 1-year isolate Q23-17 to resting peripheral blood mononuclear cells (PBMC). In contrast, resting PBMC exposed to DC pulsed with Q23ScB, which was detected infrequently in samples after seroconversion, or the 27-month chimeras were inconsistently infected. Additionally, quiescent PBMC infected with Q23ScA or Q23-17 proliferated more robustly than uninfected cells or cells infected with the other envelope chimeras in response to immobilized anti-CD3. Stimulation with tetanus toxoid led to an increased proportion of CD45RA+cells and a decreased expression of CD28 on CD45RO+cells in cultures of Q23-17-infected PBMC. These data demonstrate that variants from the heterogeneous seroconversion clade A HIV-1 population in a Kenyan woman have distinct biological features that may influence viral pathogenesis.
Measles Virus Spread by Cell-Cell Contacts: Uncoupling of Contact-Mediated Receptor (CD46) Downregulation from Virus Uptake
Authors: Firsching Ruth , Buchholz Christian J. , Schneider Urs , Cattaneo Roberto , ter Meulen Volker , Schneider-Schaulies Jürgen 
Published: December 2019

ABSTRACTCD46, which serves as a receptor for measles virus (MV; strain Edmonston), is rapidly downregulated from the cell surface after contact with viral particles or infected cells. We show here that the same two CD46 complement control protein (CCP) domains responsible for primary MV attachment mediate its downregulation. Optimal downregulation efficiency was obtained with CD46 recombinants containing CCP domains 1 and 2, whereas CCP 1, alone and duplicated, induced a slight downregulation. Using persistently infected monocytic/promyelocytic U937 cells which release very small amounts of infectious virus, and uninfected HeLa cells as contact partners, we then showed that during contact the formation of CD46-containing patches and caps precedes CD46 internalization. Nevertheless, neither substances inhibiting capping nor the fusion-inhibiting peptide Z-d-Phe-l-Phe-Gly-OH (FIP) blocked CD46 downregulation. Thus, CD46 downregulation can be uncoupled from fusion and subsequent virus uptake. Interestingly, in that system cell-cell contacts lead to a remarkably efficient infection of the target cells which is only partially inhibited by FIP. The finding that the contact of an infected with uninfected cells results in transfer of infectious viral material without significant (complete) fusion of the donor with the recipient cell suggests that microfusion events and/or FIP-independent mechanisms may mediate the transfer of MV infectivity from cell to cell.
Characterization of an Equine Arteritis Virus Replicase Mutant Defective in Subgenomic mRNA Synthesis
Authors: van Marle Guido , van Dinten Leonie C. , Spaan Willy J. M. , Luytjes Willem , Snijder Eric J. 
Published: December 2019

ABSTRACT
Equine arteritis virus (EAV) is a positive-stranded RNA virus that synthesizes a 5′- and 3′-coterminal nested set of six subgenomic mRNAs. These mRNAs all contain a common leader sequence which is derived from the 5′ end of the genome. Subgenomic mRNA transcription and genome replication are directed by the viral replicase, which is expressed in the form of two polyproteins and subsequently processed into smaller nonstructural proteins (nsps). During the recent construction of an EAV infectious cDNA clone (pEAV030 [L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991–996, 1997]), a mutant cDNA clone (pEAV030F) which carries a single replicase point mutation was obtained. This substitution (Ser-2429→Pro) is located in the nsp10 subunit and renders the EAV030F virus deficient in subgenomic mRNA synthesis. To obtain more insight into the role of nsp10 in transcription and the nature of the transcriptional defect, we have now analyzed the EAV030F mutant in considerable detail. The Ser-2429→Pro mutation does not affect the proteolytic processing of the replicase but apparently affects the function of nsp10 in transcription. Furthermore, our study showed that EAV030F still produces subgenomic positive and negative strands, albeit at a very low level. Both subgenomic positive-strand synthesis and negative-strand synthesis are equally affected by the Ser-2429→Pro mutation, suggesting that nsp10 plays an important role in an early step of EAV mRNA transcription.
Immunogenicity of a Human Immunodeficiency Virus (HIV) Polytope Vaccine Containing Multiple HLA A2 HIV CD8+Cytotoxic T-Cell Epitopes
Authors: Woodberry T. , Gardner J. , Mateo L. , Eisen D. , Medveczky J. , Ramshaw I. A. , Thomson S. A. , Ffrench R. A. , Elliott S. L. , Firat H. , Lemonnier F. A. , Suhrbier A. 
Published: December 2019

ABSTRACTCompelling evidence now suggests that αβ CD8 cytotoxic T lymphocytes (CTL) have an important role in preventing human immunodeficiency virus (HIV) infection and/or slowing progression to AIDS. Here, we describe an HIV type 1 CTL polyepitope, or polytope, vaccine comprising seven contiguous minimal HLA A2-restricted CD8 CTL epitopes conjoined in a single artificial construct. Epitope-specific CTL lines derived from HIV-infected individuals were able to recognize every epitope within the construct, and HLA A2-transgenic mice immunized with a recombinant virus vaccine coding for the HIV polytope also generated CTL specific for different epitopes. Each epitope in the polytope construct was therefore processed and presented, illustrating the feasibility of the polytope approach for HIV vaccine design. By simultaneously inducing CTL specific for different epitopes, an HIV polytope vaccine might generate activity against multiple challenge isolates and/or preempt the formation of CTL escape mutants.
p53-Independent and -Dependent Requirements for E1B-55K in Adenovirus Type 5 Replication
Authors: Harada Josephine N. , Berk Arnold J. 
Published: December 2019

ABSTRACT

The adenovirus type 5 mutant
dl
1520 was engineered previously to be completely defective for E1B-55K functions. Recently, this mutant (also known as ONYX-015) has been suggested to replicate preferentially in p53
−
and some p53
+
tumor cell lines but to be attenuated in primary cultured cells (C. Heise, A. Sampson-Johannes, A. Williams, F. McCormick, D. D. F. Hoff, and D. H. Kirn, Nat. Med. 3:639–645, 1997). It has been suggested that
dl
1520 might be used as a “magic bullet” that could selectively lyse tumor cells without harm to normal tissues. However, we report here that
dl
1520 replication is independent of p53 genotype and occurs efficiently in some primary cultured human cells, indicating that the mutant virus does not possess a tumor selectivity. Although it was not the sole host range determinant, p53 function did reduce
dl
1520 replication when analyzed in a cell line expressing temperature-sensitive p53 (H1299-tsp53) (K. L. Fries, W. E. Miller, and N. Raab-Traub, J. Virol. 70:8653–8659, 1996). As found earlier for other E1B-55K mutants in HeLa cells (Y. Ho, R. Galos, and J. Williams, Virology 122:109–124, 1982),
dl
1520 replication was temperature dependent in H1299 cells. When p53 function was restored at low temperature in H1299-tsp53 cells, it imposed a modest defect in viral DNA replication and accumulation of late viral cytoplasmic mRNA. However, in both H1299 and H1299-tsp53 cells, the defect in late viral protein synthesis appeared to be much greater than could be accounted for by the modest defects in late viral mRNA levels. We therefore propose that in addition to countering p53 function and modulating viral and cellular mRNA nuclear transport, E1B-55K also stimulates late viral mRNA translation.
Identification of the RNA-Binding, Dimerization, and eIF4GI-Binding Domains of Rotavirus Nonstructural Protein NSP3
Authors: Piron Maria , Delaunay Thierry , Grosclaude Jeanne , Poncet Didier 
Published: December 2019

ABSTRACT

The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3′ end of the rotavirus mRNAs. NSP3 also interacts with the translation initiation factor eIF4GI and competes with the poly(A) binding protein. Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in
Escherichia coli
, indicate that the RNA binding domain lies between amino acids 4 and 149. Similar results were obtained with NSP3 from group C rotaviruses. Data also indicate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding. The dimerization domain of NSP3 was mapped between amino acids 150 and 206 by using the yeast two-hybrid system. The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of NSP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system. NSP3 is composed of two functional domains separated by a dimerization domain.
Isolation of Recombinant Adeno-Associated Virus Vector-Cellular DNA Junctions from Mouse Liver
Authors: Nakai Hiroyuki , Iwaki Yuichi , Kay Mark A. , Couto Linda B. 
Published: December 2019

ABSTRACT
Recombinant adeno-associated virus (rAAV) vectors allow for sustained expression of transgene products from mouse liver following a single portal vein administration. Here a rAAV vector expressing human coagulation factor F.IX (hF.IX), AAV-EF1α-F.IX (hF.IX expression was controlled by the human elongation factor 1α [EF1α] enhancer-promoter) was injected into mice via the portal vein or tail vein, or directly into the liver parenchyma, and the forms of rAAV vector DNA extracted from the liver were analyzed. Southern blot analyses suggested that rAAV vector integrated into the host genome, forming mainly head-to-tail concatemers with occasional deletions of the inverted terminal repeats (ITRs) and their flanking sequences. To further confirm vector integration, we developed a shuttle vector system and isolated and sequenced rAAV vector-cellular DNA junctions from transduced mouse livers. Analysis of 18 junctions revealed various rearrangements, including ITR deletions and amplifications of the vector and cellular DNA sequences. The breakpoints of the vector were mostly located within the ITRs, and cellular DNA sequences were recombined with the vector genome in a nonhomologous manner. Two rAAV-targeted DNA sequences were identified as the mouse rRNA gene and the α1 collagen gene. These observations serve as direct evidence of rAAV integration into the host genome of mouse liver and allow us to begin to elucidate the mechanisms involved in rAAV integration into tissues in vivo.
Use of Major Histocompatibility Complex Class I/Peptide/β2M Tetramers To Quantitate CD8+Cytotoxic T Lymphocytes Specific for Dominant and Nondominant Viral Epitopes in Simian-Human Immunodeficiency Virus-Infected Rhesus Monkeys
Authors: Egan Michael A. , Kuroda Marcelo J. , Voss Gerald , Schmitz Jörn E. , Charini William A. , Lord Carol I. , Forman Meryl A. , Letvin Norman L. 
Published: December 2019

ABSTRACTTo evaluate the impact of the diversity of antigen recognition by T lymphocytes on disease pathogenesis, we must be able to identify and analyze simultaneously cytotoxic T-lymphocyte (CTL) responses specific for multiple viral epitopes. Many of the studies of the role of CD8+CTLs in AIDS pathogenesis have been done with simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys. These studies have frequently made use of the well-defined SIV Gag CTL epitope p11C,C-M presented to CTL by the HLA-A homologue molecule Mamu-A*01. In the present study we identified and fine mapped two novel Mamu-A*01-restricted CTL epitopes: the SIVmac Pol-derived epitope p68A (STPPLVRLV) and the human immunodeficiency virus type 1 (HIV-1) Env-derived p41A epitope (YAPPISGQI). The frequency of CD8+CTLs specific for the p11C,C-M, p68A, and p41A epitopes was quantitated in the same animals with a panel of tetrameric Mamu-A*01/peptide/β2m complexes. All SHIV-infected Mamu-A*01+rhesus monkeys tested had a high frequency of SIVmac Gag-specific CTLs to the p11C,C-M epitope. In contrast, only a fraction of the monkeys tested had detectable CTLs specific for the SIVmac Pol p68A and HIV-1 Env p41A epitopes, and these responses were detected at very low frequencies. Thus, the p11C,C-M-specific CD8+CTL response is dominant and the p41A- and p68A-specific CD8+CTL responses are nondominant. These results indicate that CD8+CTL responses to dominant CTL epitopes can be readily quantitated with the tetramer technology; however, CD8+CTL responses to nondominant epitopes, due to the low frequency of these epitope-specific cells, may be difficult to detect and quantitate by this approach.