pinecone/core-2020-05-10-deduplication huggingface dataset:
Resistance to Geminivirus Infection by Virus-Induced Expression of Dianthin in Transgenic Plants

AbstractRibosome-inactivating proteins (RIPs) are naturally occurring plant toxins that exhibit antiviral activity against a diverse range of plant and animal viruses. Here, the action of dianthin, a potent RIP isolated fromDianthus caryophyllus,has been exploited to engineer resistance to a plant DNA virus, African cassava mosaic virus (ACMV), in transgenicNicotiana benthamiana.To achieve this, dianthin has been expressed from the ACMV virion-sense promoter that is transactivated by the product of viral gene AC2. This avoids the need for constitutive expression of the RIP, facilitating the regeneration of phenotypically normal plants, and ensures transgene expression is localized to virus-infected cells. When challenged with ACMV, transgenic plants produce atypical necrotic lesions on inoculated leaves, indicative of dianthin expression, viral DNA accumulation is significantly reduced in these tissues, and plants exhibit attenuated systemic symptoms from which they recover. This phenotype holds for isolates of ACMV but not for other geminiviruses, suggesting that AC2 homologues from the latter are unable to efficiently transactivate the ACMV promoter
Construction, Expression, and Immunogenicity of Chimeric HIV-1 Virus-like Particles

AbstractThe group-specific antigens Pr55gagof human immunodeficiency virus type-1 (HIV-1) self-assemble into noninfectious virus-like particles (VLP) that are released from various eucaryotic cells by budding. Deletion analysis of Pr55gagmutants revealed three domains into which sequences of the third variable domain V3 or the CD4-binding domain of the gp120 external glycoprotein can be inserted without destroying the capacity of the chimeric proteins to assemble to VLP. Immunization of rabbits with different types of purified chimeric VLP without adjuvants raised a strong antibody response to the Pr55gagcarrier component. The magnitude of the antibody response to the inserted gp120 epitopes strictly depended on their position within thegagpolyprotein. These antisera exhibited only weak neutralizing activity. However, BALB/c mice immunized by different routes with different types of chimeric Pr55gag/V3 VLP without adjuvants developed a strong MHC class I (Dd)-restricted, cytolytic CD8+T-cell (CTL) reactivity against a known epitope within the V3 domain. When the recombinant antigen was emulsified in mineral oil (incomplete Freund's adjuvant) or adsorbed in aluminium hydroxide, its immunogenicity for CTL was drastically reduced or completely abrogated. The magnitude of the V3-specific CTL response was not influenced by the position of the V3 domain within the Pr55gag-carrier moiety; the flanking residues, hence, did not influence processing of the exogenous antigen for MHC class I-restricted peptide presentation. These results indicate ways for the rational design and optimal delivery of CTL-stimulating HIV candidate vaccines
The Human Immunodeficiency Virus Type 1 Vpu Protein Tethered to the CD4 Extracellular Domain Is Localized to the Plasma Membrane and Is Biologically Active in the Secretory Pathway of Mammalian Cells: Implications for the Mechanisms of Vpu Function

AbstractThe HIV-1 Vpu protein induces the proteolysis of CD4 in the endoplasmic reticulum (ER) and enhances the release of virus particles from the plasma membrane. The two biological activities of HIV-1 Vpu appear to be reconstituted in distinct membrane compartments of the mammalian cell. We carried out experiments to understand the role of Vpu sequences in membrane trafficking of the Vpu protein and to gain insights into Vpu-mediated proteolytic reactions. To this end, we generated CD4/Vpu hybrid proteins and analyzed their biochemical and biological properties in HeLa cells. We show here that all hybrid proteins are delivered to the plasma membrane undergoing endo-H-resistant modifications in the Golgi complex. Importantly, a hybrid protein bearing the CD4 extracellular domain and full-length Vpu induced the degradation of HIV envelope glycoproteins bearing the transmembrane and cytoplasmic domains of CD4 (Vpu-responsive elements, VRE). Glycoproteins lacking the VRE are stable under these conditions. In addition, a hybrid protein having the extracellular–transmembrane domains of CD4 and the Vpu cytoplasmic domain was only partially active in inducing the degradation of Vpu-sensitive proteins. These results suggest that the Vpu transmembrane domain is capable of regulating Vpu activity in the cell. Mutational studies have further demonstrated that casein kinase-2 phosphorylation is critically important in the degradation reaction, but does not regulate membrane trafficking of the CD4/Vpu hybrid proteins. We also show that the CD4 extracellular domain appended to the Vpu protein is protected from degradation while existing in a complex with Vpu-sensitive ectodomains. Taken together, these studies have revealed that the Vpu protein does not possess sequences that have the ability to sequester CD4 in the intracellular compartments of mammalian cells and that the Vpu protein tethered to the CD4 extracellular domain was biologically active in inducing the degradation of VRE-bearing glycoproteins in the ER
The E3-11.6-kDa Adenovirus Death Protein (ADP) Is Required for Efficient Cell Death: Characterization of Cells Infected withadpMutants

AbstractWe have reported that an 11,600-Da nuclear membrane glycoprotein named adenovirus death protein (ADP), encoded by the E3 region, is required for the efficient death (lysis) of adenovirus (Ad)-infected cells. We postulated that ADP mediates the release of virions from cells at the conclusion of replication. Here we provide further characterization of cells infected byadp+andadp−Ads. Using virus mutants with deletions in the individual E3 genes, we show that only mutants that lack ADP have small plaques that are slow to develop. Mutants in theadpgene replicated as well as wild-type Ad, but the cells lysed much more slowly. Cell lysis and viability were determined by plaque size, cell morphology, trypan blue exclusion, the release of lactate dehydrogenase, and the MTT assay for mitochondrial activity. ADP is required for efficient lysis of human A549, KB, 293, and MCF-7 cells. A549 cells infected withadp+Ads began to die at 2–3 days postinfection and were dead by 6 days. Withadpmutants, >80% of cells remained viable for 5–6 days; when the medium was changed, >80% of cells were viable after 7 days and 10–20% after 14 days. When the MTT assay was used, there was an increase in mitochondrial activity, suggesting that Ad infection stimulates respiratory metabolism. Nearly all nuclei from wild-type Ad-infected cells lacked DAPI-stained DNA by 7 days, whereas with anadpmutant nearly all nuclei stained brightly after 15 days. Nuclei fromadpmutant-infected cells were extremely swollen and full of virus, and appeared to have an intact nuclear membrane. Cells infected with wild-type Ad had many vacuoles and perhaps a disrupted nuclear membrane; they did not display features typical of apoptosis
Cis-Preferential Stimulation of Alfalfa Mosaic Virus RNA 3 Accumulation by the Viral Coat Protein

AbstractRNA 3 of alfalfa mosaic virus (AlMV) encodes the movement protein P3 and the viral coat protein (CP) which is translated from the subgenomic RNA 4. RNA 3 is able to replicate in tobacco plants transformed with the AlMV replicase genes P1 and P2 (P12 plants). Frameshifts or deletions in the P3 gene have little effect on RNA 3 accumulation in P12 protoplasts whereas such mutations in the CP gene result in a 100-fold reduction of plus-strand RNA 3 accumulation. When P12 protoplasts were inoculated with a mixture of a RNA 3 mutant with a deletion in the P3 gene and a mutant with a deletion in the CP gene, CP expressed by the P3 mutant was unable to upregulate plus-strand RNA accumulation of the CP mutant. However, when a wild-type CP gene and subgenomic promoter were inserted in a RNA 3 mutant with a defective CP gene, the mutant accumulated at wild-type levels. It is concluded that the function of CP in plus-strand RNA 3 accumulation actsin cisand cannot be complementedin trans.In P12 plants, P3 and CP mutants were able to complement each other at low and variable levels. This complementation in plants appeared to be correlated with the occurrence of recombination to wild-type RNA 3
Characterization of Human Papillomavirus-11 mRNAs Expressed in the Context of Autonomously Replicating Viral Genomes

AbstractWe have previously adapted an experimental system which supports autonomous replication of human papillomavirus-11 (HPV-11) genomic DNA in a human squamous carcinoma cell line (SCC-4) following electroporation of linearized viral DNA. This system allows evaluation of HPV-11 transcription in the context of replicating viral genomes. RNA was isolated from cells following transfection, and first-strand cDNA was produced by reverse transcription using HPV-11-specific primers. The cDNAs were amplified using the polymerase chain reaction, and resulting DNA products were cloned and sequenced. Transcripts were identified that utilize the same splice donor and acceptor sites as transcripts characterized previously from human lesions. Three previously unreported splice junctions that employ novel combinations of those splice sites were also identified. We also detected these newly identified transcripts in laryngeal papillomas. A modified version of the 5′ rapid amplification of cDNA ends method was used to identify transcriptional start sites of several of the HPV-11 transcripts. The family of mRNAs characterized in this replication system have the potential to encode all of the major HPV-11 E-region proteins described to date. The data support the utility of this system as an experimental model for examining mechanisms of viral replication and transcription
Unique Genome Arrangement of an Ovine Adenovirus: Identification of New Proteins and Proteinase Cleavage Sites

AbstractThe completed sequence and genome organization of OAV287, a serologically distinct ovine adenovirus, is described. The genome of 29,544 bp has inverted terminal repeats that are only 46 bp in length. Many OAV genes are identified by their homology with other adenovirus (Ad) sequences but three groups of reading frames show little homology. One group at the left-hand end of the genome probably represents the E1A/E1B regions. Two others, on the complementary strand at the right-hand end of the genome, are tentatively proposed as the E4 and E3 regions. They are separated by ∼1 kb of A/T-rich sequence of unknown function with E3 being adjacent to the terminus. Structural proteins V and IX of human Ads are absent from the OAV genome but a new, processed, 28-kDa virion polypeptide is encoded on the strand complementary to the proposed E1A region. The coding sequences for two other structural proteins are unidentified. The OAV penton protein lacks the region containing an Arg/Gly/Asp sequence that, in human adenoviruses, is thought to interact with cellular integrins to facilitate virus entry. Analysis of proteins and peptides in purified OAV identified several cleavage sites utilized by the Ad proteinase. Some of these were previously identified in human Ad proteins, but new sites, some of which did not conform to the known specificity of the human Ad proteinase, were also identified. The data emphasize that this ovine virus differs significantly from other known human and animal adenoviruses
Construction and Transfection of Ovine Adenovirus Genomic Clones to Rescue Modified Viruses

AbstractThe genome of ovine adenovirus OAV287 has an arrangement which is unique among known adenoviruses. To facilitate further experimentation on the structure and function of this genome, plasmids containing a complete clone of the genome were constructed. The cloned viral genome was released from plasmids by restriction enzyme digestion as an intact linear molecule with authentic 5′ termini. Transfection of the linear DNA into cells which supported replication produced infectious virus. Mutation of a uniqueSalI site at the right-hand end of the genome disrupted reading frames of unknown function without affecting virus rescue, identifying this region as nonessential for replicationin vitro.A 20-bp oligonucleotide was also inserted into the short intergenic region between the pVIII and the fiber sequences, identifying a second site for gene insertion. These studies will facilitate the development of OAV as a gene transfer vector
The E1 Protein Is Mandatory for Pore Formation by Semliki Forest Virus Spikes

AbstractInsect cells (Aedes albopictus,clone C6/36) were infected with various variants of Semliki Forest virus including the wild type using the SFV replicon system. The variants included deletion mutants lacking one of the structural proteins and a mutant with a point mutation in p62 (SQL). The latter mutation results in a failure to process p62 to E2 and E3. After infection of the cells with different variant viruses and subsequent expression of viral proteins in the host cell plasma membrane low pH-induced pore formation was detected by measuring the efflux of a radiolabeled compound. The results of these experiments clearly showed that the E1 protein is mandatory for the acid-induced pore formation. A participation of the 6K or C-protein could be excluded. Furthermore, results obtained with the SQL mutant suggest that dissociation of the E1/E2 heterodimer and subsequent homooligomerization of E1 are required for pore formation
Evidence for Direct Association of Vpr and Matrix Protein p17 within the HIV-1 Virion

AbstractVpr is one of the auxiliary proteins of HIV-1 and is selectively incorporated into the virion by a process involving the C-terminal p6 portion of the Gag precursor Pr55. Vpr and the matrix protein p17 are the components of the viral preintegration complex and appear to play important roles in the nuclear transport of proviral DNA in nondividing cells. In the present study, we have demonstrated by coimmunoprecipitation experiments that Vpr associates with matrix protein p17 but not with capsid protein p24 within the HIV-1 virion. Experiments employing the yeast two-hybrid GAL4 assay for protein–protein interactions also demonstrated a direct association between Vpr and the C-terminal region of matrix protein p17. Association of Vpr and the matrix protein p17 within the mature virion is consistent with their collaborative role in the nuclear transportation of the viral preintegration complex in nondividing cells such as macrophages
Gain of Sp1 Sites and Loss of Repressor Sequences Associated with a Young, Transcriptionally Active Subset of HERV-H Endogenous Long Terminal Repeats

AbstractHERV-H sequences comprise a large family of human endogenous retrovirus-like elements. Previous DNA sequence comparisons of HERV-H long terminal repeats (LTRs) have led to their classification into three subtypes, Types I, Ia, and II. Type Ia appears to have been generated by recombination between Type I and Type II LTRs. These subtypes differ in evolutionary age and transcriptional activity with Type Ia LTRs being younger in evolutionary terms and possessing stronger promoter function than the other two subtypes. In this study, possible mechanisms responsible for the functional difference between LTRs have been explored. Types I and II LTRs each contain different sets of repeated segments in their U3 regions which are disrupted in Type Ia LTRs. Using reporter gene assays, we have shown that both types of repeated segments can suppress activity of the human β-globin gene promoter when cloned at a distant site. Both sets of repeats also repress promoter activity of a Type Ia LTR when directly inserted within its U3 region. In addition, using deletion constructs, we have localized two positive regulatory segments within the Type Ia LTR, both of which contain a potential binding site for the transcription factor Sp1. Gel mobility shift assays demonstrated that fragments containing these sites do bind Sp1. Although Type I LTRs are generally similar to Type Ia LTRs in the regions surrounding the Sp1 sites, there are sequence differences within the sites. Gel-shift analysis revealed no or much reduced Sp1 binding of Type I LTR fragments containing these sites. Thus, it appears that the loss of repeated suppresser elements and the acquisition of Sp1-binding sites have both contributed to the relatively strong transcriptional activity of the Type Ia LTRs
Immunization with VP2 Is Sufficient for Protection against Lethal Challenge with African Horsesickness Virus Type 4

AbstractHorses were immunized by inoculation with a vaccinia construct containing a full-length cDNA corresponding to the L2 gene segment of African horsesickness virus type 4 (AHSV-4). All immunized horses developed serum neutralizing antibodies prior to challenge with virulent AHSV-4. No ELISA-reactive antibodies were present prior to challenge. A group of four seronegative control horses died after developing clinical signs and lesions typical of the pulmonary form of African horsesickness while the immunized horses were clinically normal. Increases in serum neutralizing and ELISA-reactive antibody titers following challenge indicate that at least some replication of challenge virus occurred in immunized horses. These results demonstrate that AHSV VP2 alone is sufficient to induce a protective immune response in horses and indicate the usefulness of ELISA-reactive antibodies for differentiation of vaccinated and naturally exposed horses
Expression of the Major Core Structural Protein (VP7) of Bluetongue Virus, by a Recombinant Capripox Virus, Provides Partial Protection of Sheep against a Virulent Heterotypic Bluetongue Virus Challenge

AbstractA recombinant capripox virus was constructed containing a cDNA copy of genome segment 7 of bluetongue virus (BTV) serotype 1 from South Africa (BTV 1SA), which expressed high levels of the major BTV core protein VP7 in infected lamb testis (LT) cells. Sheep vaccinated with this recombinant virus developed antibodies to VP7 (detected by ELISA) but no neutralizing antibodies to either the homologous or heterologous BTV serotype, prior to challenge (BTV 1 or BTV 3, respectively). Following challenge with a virulent heterotypic strain of BTV (BTV3 SA), all of the animals developed clinical signs of disease, indicating that they were infected and that the challenge virus did replicate. While all of the control animals died, six of the eight animals that were vaccinated with the recombinant capripox virus expressing VP7 recovered fully. This is the first report of a significant level of cross serotype protection against the lethal effects of a challenge with virulent BTV, produced by vaccination with a single BTV core protein, which did not generate a neutralizing antibody response
Immunolocalization of the Dengue Virus Nonstructural Glycoprotein NS1 Suggests a Role in Viral RNA Replication

AbstractAlthough many workers have investigated the maturation and processing of the flavivirus nonstructural glycoprotein NS1 in infected cells, these studies have provided little insight into a possible function for NS1. In this study we investigated the subcellular localization of NS1 both by immunofluorescence and cryo-immuno electron microscopy of infected Vero and C6/36 cells. NS1 was found to be tightly associated with intracellular membranes, in particular with vesicle packets and large cytoplasmic vacuoles. Surprisingly, NS1 did not associate with mature virus particles, a finding that is inconsistent with the postulated role for this protein in virion assembly and/or maturation. However, dual-labeling experiments did reveal in both the confocal immunofluorescence and cryo-immuno EM studies the colocalization of NS1 with the viral dsRNA replicative form. Furthermore, localization of the dsRNA to the vesicle packets and cytoplasmic vacuoles seen in infected Vero and C6/36 cells, respectively, suggests that these structures may comprise the flavivirus replication complex. These findings provide further insights into the maturation pathway of NS1 and suggest a possible role for this protein in viral RNA replication
Expression of the Human MxA Protein Is Associated with Hyperphosphorylation of VSV P Protein in Human Neural Cells

AbstractConstitutive expression of the type I interferon-inducible human cytoplasmic MxA protein has been shown to interfere with primary transcription of vesicular stomatitis virus (VSV) in tissue culture cells. As phosphorylation of the VSV P protein has been linked to its ability to stimulate viral transcription, we analyzed the phosphorylation status of this protein in human brain cells (U-87) stably transfected with MxA. We observed a general increase in cellular kinase activity in the presence of MxA, affecting both cellular proteins and VSV P protein. Phosphorylation of the latter was up to threefold higher bothin vivoandin vitro. In vitrophosphorylation of recombinant VSV P protein could be enhanced in MxA-negative cell extracts after exogenous addition of recombinant His-MxA. Biochemical evidence and phosphorylation of a mutant P protein lacking the recognized casein kinase II (CKII) sites suggested that hyperphosphorylation of VSV P protein was not due to a stimulation of CKII. We thus propose that expression of MxA in human brain cells is associated with the stimulation of a cellular kinase that is active in phosphorylating both cellular target proteins and VSV P protein
The Structure of a Highly Virulent Theiler's Murine Encephalomyelitis Virus (GDVII) and Implications for Determinants of Viral Persistence

AbstractGDVII is a highly virulent Theiler's murine encephalomyelitis virus (TMEV) which causes acute encephalitis in mice, while the BeAn and DA strains are the less virulent TMEV which cause chronic demyelinating disease in the central nervous system as a result of persistent infection. Purified GDVII virus isolated from infected BHK-21 cells was crystallized and its structure was determined to 3.5-Å resolution by X-ray crystallography. In contrast to other TMEV structures, the VP1 C-terminus of GDVII virus has an ordered conformation that forms a hook over the VP3 knob near the threefold axis. Comparisons with the atomic structures of the less virulent BeAn and DA viruses revealed significant structural variations in a major site (cluster B) on the protruding surface loop puff B of VP2. Puff B is located near the VP3 GH loop region which is structurally analogous to the host receptor attachment site of the major serogroup of human rhinoviruses. Mutations at residue 1101 in VP1 and residue 2141 in VP2, which are also near the VP3 GH loop and adjacent to cluster B, were previously shown to influence persistence of DA virus. These observations indicate that the characteristic interaction with the host receptor through these sites may potentially alter TMEV persistence
Characterization of the Human Cytomegalovirus UL105 Gene and Identification of the Putative Helicase Protein

AbstractWe have characterized the transcription unit for the human cytomegalovirus UL105 gene and identified its putative protein product. The UL105 gene product is proposed to mediate helicase activity in the assembled helicase–primase complex. The two other putative proteins in this complex are the gene products of UL102 (primase-associated factor) and UL70 (primase). Using Northern blot analysis we have determined that the UL105 transcript is a 3.4-kb message that can be detected as early as 24 hr postinfection in the presence of phosphonoformic acid but not in the presence of cycloheximide. Subsequent primer-extension analysis showed a transcriptional start site upstream of a consensus TATA sequence and just downstream of a CCAAT box sequence motif. In addition, we have identified an infected cell protein with an approximate molecular weight (Mr) of 110 kDa using anti-peptide antiserum. This same antiserum detected proteins of the sameMras those produced from two expression systems where the protein from the 981-amino-acid UL105 open reading frame was overexpressed, suggesting that the ATG located at nt 151,850 of the genomic sequence is utilized in the context of the virus genome