pinecone/core-2020-05-10-deduplication huggingface dataset:
Rapid Activation of Poly(ADP-ribose) Polymerase Contributes to Sindbis Virus and Staurosporine-Induced Apoptotic Cell Death

AbstractPoly(ADP-ribose) polymerase-1 (PARP-1) is a chromatin-associated enzyme that is activated by DNA strand breaks and catalyzes the transfer of ADP-ribose groups from NAD to itself and other nuclear proteins. Although caspase-mediated PARP-1 cleavage occurs during almost all forms of apoptosis, the contribution of PARP-1 activation and cleavage to this cell death process remains unclear. Using immortalized fibroblasts from wild-type (PARP-1+/+) and PARP-1 knockout (PARP-1−/−) mice, and a mouse neuroblastoma cell line (N18), the role that poly(ADP-ribosyl)ation plays in Sindbis virus (SV)-induced apoptosis was examined. Robust PARP-1 activation occurred in SV-infected cells prior to morphologic changes associated with apoptotic cell death and PARP-1 activity ceased simultaneously with caspase-3 activation and PARP-1 proteolysis. PARP-1 activity was maximal before detectable DNA fragmentation, but was absent when DNA damage was most intense. SV and staurosporine-induced cell death was delayed in fibroblasts lacking PARP-1 activity, suggesting that PARP-1 activation contributes to apoptotic cell death induced by these stimuli. SV replication was not affected by lack of PARP-1 activity, but DNA fragmentation and caspase-3 activation were delayed and occurred at lower levels in PARP-1-deficient fibroblasts. Early virus-induced PARP-1 activation may represent a novel way by which cells signal to the nucleus to regulate protein function by poly(ADP-ribosyl)ation in response to virus infection
Uptake and Processing of Modified Bacteriophage M13 in Mice: Implications for Phage Display

AbstractInternalization and degradation of filamentous bacteriophage M13 by a specific target cell may have major consequences for the recovery of phage in in vivo biopanning of phage libraries. Therefore, we investigated the pharmacokinetics and processing of native and receptor-targeted phage in mice. 35S-radiolabeled M13 was chemically modified by conjugation of either galactose (lacM13) or succinic acid groups (sucM13) to the coat protein of the phage to stimulate uptake by galactose recognizing hepatic receptors and scavenger receptors, respectively. Receptor-mediated endocytosis of modified phage reduced the plasma half-life of native M13 (t1/2 = 4.5 h) to 18 min for lactosylated and 1.5 min for succinylated bacterophage. Internalization of sucM13 was complete within 30 min after injection and resulted in up to 5000-fold reduction of bioactive phage within 90 min. In conclusion, these data provide information on the in vivo behavior of wild-type and receptor-targeted M13, which has important implications for future in vivo phage display experiments and for the potential use of M13 as a viral gene delivery vehicle
Human Cytotoxic T Lymphocyte Responses to Live Attenuated 17D Yellow Fever Vaccine: Identification of HLA-B35-Restricted CTL Epitopes on Nonstructural Proteins NS1, NS2b, NS3, and the Structural Protein E

AbstractYellow fever virus (YFV) is a reemerging problem despite the existence of an effective live-attenuated vaccine. The induction of YFV-neutralizing antibodies undoubtedly contributes to vaccine efficacy, but T lymphocyte responses to YFV likely play a role in long-term efficacy. We studied the T lymphocyte responses to YFV in four vaccinees. Proliferation and cytolytic responses to YFV were demonstrated in all subjects. We isolated 13 YFV-specific CD8+ CTL lines that recognized epitopes on the E, NS1, NS2b, and NS3 proteins; eight CTL lines were HLA-B35-restricted. YFV-specific T cell responses were detectable by IFNγ ELISPOT assays 14 days postvaccination, with T cell frequencies sustained for up to 19 months. To our knowledge, this is the first report of human T lymphocyte responses following YFV vaccination. These results indicate that the live 17D YFV vaccine induced CD8+ T cell responses directed against at least four different HLA-B35-restricted YFV epitopes
Adenoassociated Virus Type 2-Induced Inhibition of the Human Papillomavirus Type 18 Promoter in Transgenic Mice

AbstractThe epithelium of the cervix uteri has been reported to be frequently coinfected with both human papillomaviruses (HPV) and helper virus-dependent adenoassociated viruses (AAV). Seroepidemiological data suggest that AAV infection could inhibit cervical cancer that is caused by specific (“high-risk”) types of papillomaviruses. In vitro, infection with AAV type 2 (AAV-2) or transfection of AAV-2 early (rep) genes has been shown to inhibit transformation by papillomaviruses. To analyze the effects of AAV on HPV in vivo, we studied the influence of AAV-2 infection on the promoter activity of high-risk HPV type 18 (HPV-18) in mice, transgenic for sequences of the upstream regulatory region (URR) of HPV-18 controlling transcription of the reporter gene, lacZ. Transgenic animals (or tongue cells thereof, explanted and grown in culture) were treated with dexamethasone to induce the HPV-18 promoter. Simultaneously they were (i) infected with AAV, (ii) inoculated with AAV virus-like particles (VLPs; empty capsids), or (iii) mock infected. Inoculation with AAV-2 or VLPs inhibited activation of the HPV-18 promoter. In vitro, in baby hamster kidney cells transfected with the HPV-18-lacZ construct, tissue extracts from AAV-infected animals suppressed the HPV-18 URR to a similar extent as AAV infection did. Down-regulation of the HPV-18 promoter was less efficient with extracts from animals inoculated with VLPs and was not observed with extracts from uninfected or dexamethasone-treated animals. This indicates that AAV induces cellular factor(s) in vivo capable of mediating down-regulation of the HPV-18 promoter also in cells in vitro. In contrast, promoters of the low-risk HPV types (HPV-6, HPV-11) were not influenced by AAV infection as opposed to promoters of the high-risk types (HPV-18 and HPV-16)
Loss of Activities for mRNA Synthesis Accompanies Loss of λ2 Spikes from Reovirus Cores: An Effect of λ2 on λ1 Shell Structure

AbstractThe 144-kDa λ2 protein, a component of the transcriptionally active reovirus core particle, catalyzes the last three enzymatic activities for formation of the 5′ cap 1 structure on the viral plus-strand transcripts. Limited evidence suggests it may also play a role in transcription per se. Particle-associated λ2 forms pentameric turrets (“spikes”) around the fivefold axes of the icosahedral core. To address the requirements for λ2 in core functions other than the known functions in RNA capping, particles depleted of λ2 were generated from cores in vitro by a series of treatments involving heat, protease, and ionic detergent. The resulting particles contained less than 5% of pretreatment levels of λ2 but showed negligible loss of the other four core proteins or the 10 double-stranded RNA genome segments. Transmission cryo-electron microscopy (cryo-TEM) and scanning cryo-electron microscopy demonstrated loss of the λ2 spikes from these otherwise intact particles. In functional analyses, the “spikeless cores” showed greatly reduced activities not only for RNA capping but also for transcription and nucleoside triphosphate hydrolysis, suggesting enzymatic or structural roles for λ2 in all these activities. Comparison of the core and spikeless core structures obtained by cryo-TEM and three-dimensional image reconstruction revealed changes in the λ1 core shell that accompany λ2 loss, most notably the elimination of small pores that span the shell near the icosahedral fivefold axes. Changes in the shell may explain the reductions in transcriptase-related activities by spikeless cores
T Cell Epitopes in Coxsackievirus B4 Structural Proteins Concentrate in Regions Conserved between Enteroviruses

AbstractThe present study aimed to characterize systematically the target epitopes of T cell responses in CBV4 structural proteins. These were studied by synthesizing 86 overlapping 20-aa-long peptides covering the known sequence of CBV4 structural proteins and analyzing the proliferation responses of 18 CBV4-specific T cell lines against these peptides. Recognized peptides differed depending on the HLA-DR genotype of the T cell donor. They were concentrated to the VP4 and VP2 regions as six of seven common peptide epitopes located in this region, whereas there was only one in the VP3 region and none in the VP1 region. Peptides from conserved areas were recognized more often (on average, 15% of them stimulated each T cell line) than those derived from variable areas (3%) (P < 0.0001, Fisher's exact test). Some conserved peptides inducing T cell responsiveness in most subjects were identified, a knowledge which can be useful in the development of new synthetic vaccines
Site-Directed Mutagenesis of Avibirnavirus VP4 Gene

AbstractVirus protein VP4 of infectious bursal disease virus (IBDV) is a protease which separates VPX and VP3 from the polyprotein. We studied the importance of serine and aspartic acid on cleavage at the VPX/VP4 junction and analysed the role of the proposed H547, D590, and S653 catalytic site using five different mutations on VP4. Our results suggest that the replacement of serine by lysine in AXAAS motifs in serotype II IBDV influences polyprotein (PP) processing by VP4 and also indicate the presence of an alternative cleavage site. Mutation on D (510TLAADK515) prevented the cleavage at the VPX/VP4 junction, but we have found that independently of the importance of those alanines in LAA, D has an important role as part of the cleavage site. Replacement of histidine by proline H547P completely abolished PP processing. Mutation on D590 induced a partial PP processing when it was replaced by proline and the replacement of serine by proline at S653P induced a prominent change in PP processing. These results permit us to conclude that IBDV VP4 has the ability to act according to structural and topographical changes during translational and posttranslational processes and allow multiple hit sites, which serve to increase effectiveness
Hypophosphorylation of Poly(A) Polymerase and Increased Polyadenylation Activity Are Associated with Human Immunodeficiency Virus Type 1 Vpr Expression

AbstractThe HIV-1 encoded accessory protein, viral protein R (Vpr) is responsible for several biological effects in HIV-1-infected cells including nuclear transport of the preintegration complex, activation of long terminal repeat (LTR)-mediated transcription, and the induction of cell-cycle arrest and apoptosis. Vpr's ability to arrest cells at the G2 phase of the cell cycle is due to the inactivation of p34cdc2 cyclin B complex, resulting in hypophosphorylation of substrates involved in cell-cycle progression from G2 to mitosis (M). Poly(A) polymerase (PAP), the enzyme responsible for poly(A) addition to primary transcripts, contains multiple consensus phosphorylation sites for p34cdc2 cyclin B kinase that regulates its catalytic activity. We investigated the effects of Vpr on the activity of PAP in Jurkat cells using a superinfection system. Superinfection of cells using Vpr+ vesicular stomatitis virus G protein (VSV-G)-pseudotyped virus caused a complete dephosphorylation of PAP. Cotransfection studies in 293T cells and Xenopus oocyte RNA injection experiments mirrored these effects. Vpr's dramatic effect on PAP dephosphorylation was reflected in enhanced polyadenylation activity in PAP activity assays. HIV-1 Vpr appears to enhance processes that are coupled to transcription such as polyadenylation and could ultimately prove to optimize HIV-1 replication and contribute to HIV-1 pathogenesis
Generation of E3-Deleted Canine Adenoviruses Expressing Canine Parvovirus Capsid by Homologous Recombination in Bacteria

AbstractE3-deleted canine adenovirus type 1 (CAV-1) was generated by homologous recombination in bacterial cells, using an antibiotic resistance marker to facilitate the recovery of recombinants. This marker was flanked by unique restriction endonuclease sites, which allowed its subsequent removal and the insertion of cassettes expressing the canine parvovirus capsid at the E3 locus. Infectious virus was recovered following transfection of canine cells and capsid expression was observed by RT-PCR from one of the virus constructs. A second construct, containing a different promoter, showed delayed growth and genome instability which, based on the size difference between these inserts, suggests a maximum packaging size of 106 to 109% wild-type genome size for CAV-1
Inhibition of Serum- and Calcium-Induced Terminal Differentiation of Human Keratinocytes by HPV 16 E6: Study of the Association with p53 Degradation, Inhibition of p53 Transactivation, and Binding to E6BP

AbstractTransfection of the E6 gene of human papillovirus (HPV) 16 into primary human keratinocytes (PHKs) generates proliferating cell colonies which are resistant to serum- and calcium-induced terminal differentiation. The extreme C-terminus of E6 was shown to be dispensable for this activity. To map further the amino acid sequences required for inducing resistance to serum and calcium, and to address the functional significance of E6 interactions with p53 and E6BP (ERC-55) in this function, we evaluated the activities of a series of E6 mutants. Small deletions within the central portion of the second putative zinc-finger abolished, or markedly reduced, E6 biological activity, while mutations affecting the cysteine residues in the base of the finger were less effective in this respect. When these mutants were assayed for their ability to degrade p53 in vitro and in vivo and to inhibit p53 transcriptional activation (TA), we found that there was a dissociation of these activities in some mutants. We mapped one mutant which was highly efficient in p53 degradation and inhibition of p53 TA, yet displayed severely reduced activity in the biological assay, and conversely, a subset of mutants that showed moderate activities in the colony assay while being severely impaired in p53 degradation and inhibition of p53 TA. These data argue that p53 inactivation or even elimination are not sufficient, and may not be essential, for altering the response of PHKs to serum and calcium. When these E6 mutants were evaluated for E6BP binding in vitro, there was a similar dissociation between the biological and biochemical activities of several mutants. We mapped mutants with moderate activity in the biological assay that lacked the ability to bind to E6BP and a mutant that showed high biological activity with only marginal binding to E6BP. Thus, there is no absolute correlation between the ability of E6 mutant proteins to induce alterations in keratinocyte differentiation responses to calcium and serum and to induce p53 degradation, inhibit p53 mediated transactivation, or bind E6BP. Evidently there are additional cellular targets for E6 which mediate this alteration in cellular differentiation
Bovine Herpesvirus 1 Glycoprotein G Is Necessary for Maintaining Cell-to-Cell Junctional Adherence among Infected Cells

AbstractGlycoproteins gE and gG of bovine herpesvirus 1 (BHV-1) are involved in viral cell-to-cell transmission. We have compared the subcellular localizations of gE and gG and examined the cell-to-cell adherence of bovine kidney (MDBK) cells infected with BHV-1 mutants lacking gE or gG. In BHV-1-infected MDBK cells, gE was observed at cell junctions but did not localize at apical or basal plasma membranes. BHV-1 gG was primarily found in the cytoplasm and was also observed at boundaries among infected cells. During the infection with wild-type or gE-negative BHV-1, the filamentous actin and the adherent junctional proteins accumulated at the cell junctions. In contrast, cell junctions of MDBK cells infected with gG-negative BHV-1 were loosened, and the junctional proteins and BHV-1 gE were distributed in the cytoplasm. These data indicate that BHV-1 gG facilitates viral cell-to-cell spread by maintaining the cell-to-cell junctions among the infected cells
The Role of the CD4 Receptor versus HIV Coreceptors in Envelope-Mediated Apoptosis in Peripheral Blood Mononuclear Cells

AbstractWe examined the role of CD4, CXCR4, and CCR5 in HIV envelope-mediated apoptosis by measuring the response of activated PBMCs to recombinant envelope proteins derived from CXCR4- and CCR5-utilizing viruses. Apoptosis of T cells was assessed by annexin-V staining and TdT-mediated dUTP-biotin nick-end labeling. Treatment of CCR5Δ32 homozygote PBMCs with a CCR5-specific envelope induced apoptosis in T cells, demonstrating that envelope–CD4 interactions are sufficient to induce apoptosis. However, a CXCR4-specific envelope induced higher levels of apoptosis than a CCR5-specific envelope, suggesting that envelope-mediated apoptosis can be enhanced by envelope–CXCR4 interactions. We conclude that envelope can induce apoptosis in T cells independently of the coreceptor specificity of a given envelope, or the expression profile of CXCR4 or CCR5 on a target cell. However, envelope–coreceptor interactions, and in particular, envelope–CXCR4 interactions, can contribute to this process
Spontaneous Mobilization of Integrated Recombinant Adenoassociated Virus in a Cell Culture Model of Virus Latency

AbstractA cell line containing integrated recombinant adenoassociated virus (AAV) was investigated for spontaneous mobilization of vector sequence. Detection of these rare events was facilitated by using a vector design that allowed the circular rescue product (cAAV) to be individually scored by bacterial transformation. Restriction and sequence analysis of captured clones revealed five highly ordered classes of cAAV, each of which contained a defined segment of the integrated vector locus. A common feature of all cAAV classes was the presence of a modified inverted terminal repeat that joined the ends of the liberated sequence. Assembly of extrachromosomal vector genomes was accompanied by deletions in the integration locus that could be mapped to one of the five cAAV classes, suggesting an excision-type mechanism. We propose that the spontaneous deletion and mobilization of vector sequence from the recombinant adenoassociated virus (rAAV) integration locus is mediated by a recombination event between the inverted terminal repeats that define the boundaries of the individual genome subunits
The 3′-Terminal Structure Required for Replication of Barley Yellow Dwarf Virus RNA Contains an Embedded 3′ End

AbstractWe determined the 3′-terminal primary and secondary structures required for replication of Barley yellow dwarf virus (BYDV) RNA in oat protoplasts. Computer predictions, nuclease probing, phylogenetic comparisons, and replication assays of specific mutants and chimeras revealed that the 3′-terminal 109 nucleotides (nt) form a structure with three to four stem-loops followed by a coaxially stacked helix incorporating the last four nt [(A/U)CCC]. Sequences upstream of the 109-nt region also contributed to RNA accumulation. The base-pairing but not the sequences or bulges in the stems were essential for replication, but any changes to the 3′-terminal helix destroyed replication. The two 3′-proximal tetraloops tolerated all changes, but the two 3′-distal tetraloops gave most efficient replication if they fit the GNRA consensus. A mutant lacking the 3′-proximal stem-loop produced elevated levels of less-than-full-length minus strands, and no (+) strand. We propose that a “pocket” structure is the origin of (−)-strand synthesis, which is negatively regulated by the inaccessible conformation of the 3′ terminus, thus favoring a high (+)/(−) ratio. This 3′ structure and the polymerase homologies suggest that genus Luteovirus is more closely related to the Tombusviridae family than to other Luteoviridae genera
Viral and Cellular Specificities of Caprine Arthritis Encephalitis Virus Vif Protein

AbstractThe caprine arthritis encephalitis virus (CAEV) Vif protein is necessary for a productive infection of susceptible goat cells. The vif gene is conserved among all primate and most nonprimate lentiviruses. However, previous reports demonstrated that, in their respective host cells, primate Vif deleted lentiviruses could not be complemented by nonprimate Vif proteins, suggesting that species-specific restrictions between Vif and the virus-producing cells may modulate the Vif function on viral infectivity. Here we bring further support to this hypothesis since we show that CAEV Vif, when expressed in goat cells, is able to increase the infectivity of Vif deleted human immunodeficiency type-1 virus and of murine leukemia virus. Moreover, we demonstrate in vitro interactions between different Vif proteins and NC domains of heterologous Gag precursors, supporting the notion that species specificity of lentiviral infection is not due to molecular interactions between Vif and viral components
Autogenous Translational Inhibition of Core Protein: Implication for Switch from Translation to RNA Replication in Hepatitis C Virus

AbstractPositive-stranded viruses use the genomic RNA as a common template for translation and RNA replication which proceed in inverse direction; a certain regulatory mechanism for translation control is probably required to coordinate these two antagonistic processes. Hepatitis C virus (HCV) core protein is a good candidate that might play a role in such a regulation. In this study, we further investigated whether HCV core protein modulates internal ribosome entry site (IRES)-directed translation. The inclusion of the core-coding sequence significantly suppressed translation initiated by HCV IRES in monocistronic and bicistronic reporter systems. The region mainly responsible for this inhibition was mapped to nt 441–473 of the core-coding sequence. This suppression was eliminated by frameshift mutations introduced into this region, suggesting that it is the core protein expressed in cis, rather than the core-coding nucleotide sequence that negatively modulates the efficiency of HCV IRES-dependent translation. Furthermore, the core protein provided in trans also specifically decreased the IRES activity in directing cap-independent translation both in transfected cells and in cell-free translation study. Consistently, a gel mobility shift assay showed a specific interaction between the core protein and HCV IRES-containing RNA transcript. These findings suggest that HCV core protein may down-regulate the cap-independent translation as a regulatory mechanism required for initiation of transcription
Genetic Constraints and the Adaptive Evolution of Rabies Virus in Nature

AbstractWe used a molecular evolutionary approach to investigate the species adaptation of rabies virus in nature. A maximum likelihood analysis of selection pressures revealed that the nucleoprotein (N) and glycoprotein (G) genes of natural viral isolates were highly constrained, especially at nonsynonymous sites, in contrast to the higher rates of nonsynonymous evolution observed in viruses subject to laboratory passage. Positive selection was only found at a single amino acid site—position 183 in the ectodomain of the G gene. The low rate of nonsynonymous evolution in natural isolates of rabies virus may be due to constraints imposed by the need to replicate in multiple cell types within the host, which in turn facilitates cross-species transmission, or because viral proteins are not subject to immune selection. Using known dates in the epidemiologic history of European viral isolates, we estimated that overall rates of nucleotide substitution in rabies virus were similar to those observed in other RNA viruses. Assuming that the average rate of synonymous change does not vary among species, we estimated that the current genetic diversity in lyssavirus genotype 1 may have arisen only during the last 500 years