pinecone/core-2020-05-10-deduplication huggingface dataset:
Sequence Analysis of the Spodoptera litura Multicapsid Nucleopolyhedrovirus Genome

AbstractThe complete Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) genome contained 139,342 bp with a G+C content of 42.7%, and 141 putative open reading frames (ORFs) or genes of 150 nucleotides or greater that showed minimal overlap. Ninety-six ORFs had homologues in Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), 16 had homologues in other baculoviruses, and 29 were unique to SpltMNPV. The homologues of ubiquitin and gp37 are fused in SpltMNPV. The genome lacked a homologue of the major budded virus glycoprotein gene gp64, but it contained a homologue of ORF130 of Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV). There were two homologues of AcMNPV ORF2 (bro gene), and a DnaJ protein gene (SpltORF39) in which the N-terminus showed homologies with the J domain of DnaJ family proteins. Seventeen homologous regions (hrs) were identified, each containing 2–29 palindromic repeats, with an average length of 534 bp and base content (G+C%) of 33.0
Properties of Two EBV Mta Nuclear Export Signal Sequences

AbstractThe Epstein–Barr virus (EBV) Mta protein is a posttranscriptional regulator of EBV lytic gene expression that affects RNA splicing and transport. Mta mediates cytoplasmic accumulation of unspliced EBV replication gene transcripts and shuttles between the nucleus and cytoplasm. Mta contains a recognized leucine-rich, putative nuclear export signal (NES) between aa 227 and 236. Deletion of this signal sequence eliminated shuttling, while mutation of the core LXL motif in the putative NES diminished but did not abolish the ability of Mta to shuttle from donor to recipient cells in a heterokaryon assay. A double mutation of the LXL motif plus an upstream VTL motif eliminated shuttling, suggesting that Mta may have two NES motifs. In confirmation of this, transfer of either the sequence encoding the leucine-rich aa 227–236 motif or that encoding the adjacent hydrophobic aa 218–227 sequence to a GFP-NLS-pyruvate kinase reporter protein conferred the property of cytoplasmic accumulation onto the heterologous protein. Cytoplasmic accumulation of both the aa 225–237 and 218–227 containing reporters was minimal in the presence of the inhibitor leptomycin B, indicating that both motifs mediated Crm-1-dependent export. Mutations in the NES signal sequences abolished the ability of Mta to mediate cytoplasmic accumulation of BALF2 replication gene transcripts. This included mutation of the LXL motif which still showed cytoplasmic shuttling, suggesting that the NES mutations might have additional effects on Mta function. Wild-type Mta co-immunoprecipitated with the splicing factor SC35 and colocalized with SC35 in transfected cells, modifying endogenous SC35 distribution within the nucleus to give more intense, rounded spots. Interestingly, the NES mutant proteins appeared to have altered interactions with the splicing complex, binding more tightly to SC35 in co-immunoprecipitation assays. These observations suggest a linkage between the splicing and export functions of Mta
Mechanisms of Loss of Foreign Gene Expression in Recombinant Vesicular Stomatitis Viruses

AbstractWe investigated the stability and mechanisms of loss of foreign gene expression in two recombinant vesicular stomatitis viruses (VSVs). A recombinant expressing the cellular CD4 protein exhibited remarkable stability of foreign gene expression. However, after 26 sequential passages, a mutant no longer expressing CD4 was recovered from the virus stock. Sequencing of the CD4 coding region in this mutant revealed a single nucleotide deletion causing a frameshift and termination of protein synthesis. A second VSV recombinant expressing the measles virus F protein grew poorly and exhibited extreme instability of expression of the F protein. Expression of F protein was lost rapidly through mutations of the upstream transcription termination site from 3′AUAC5′ to 3′AUAU5′, as well as lengthening of the subsequent U7 tract that is the template for poly(A) addition to VSV G mRNA. Such mutations resulted in fusion of the F mRNA to the 3′ end of the G mRNA, making the F protein translation initiation codon inaccessible. We suggest that the VSV polymerase is error prone during replication of the U7 tract, providing a rapid means for complete elimination of expression of proteins that are toxic to the virus life cycle
HIV-1 LTR Subtype and Perinatal Transmission

AbstractMultiple subtypes of HIV-1 have been identified; however, there is little data on the relative transmissibility of viruses belonging to different subtypes. A matched case-control study addressed whether viruses with different long terminal repeat (LTR) subtypes were transmitted equally from mother to infant. The LTR subtype was determined for 45 matched cases and controls who participated in a clinical trial in Tanzania. HIV-1 subtypes A, C, and D and intersubtype recombinant sequences were identified. Exact matched logistic regression analysis showed that viruses containing subtype A or intersubtype recombinant LTRs were 3.2 and 4.8 times more likely to be transmitted from mother to infant than viruses with subtype D LTRs. Viruses containing subtype C LTRs were 6.1 times more likely to be transmitted than those with subtype D LTRs. These differences in transmission were independent of maternal CD4 at enrollment. Thus, it appears that HIV-1 subtype may be associated with differing rates of perinatal transmission in Tanzania
Trans-Complementation of Long-Distance Movement of White clover mosaic virus Triple Gene Block (TGB) Mutants: Phloem-Associated Movement of TGBp1

AbstractThe triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Both cell-to-cell and long-distance movement of White clover mosaic virus in which individual, combinations, or all movement functions were mutated could be rescued by transgenic Nicotiana benthamiana expressing complementary viral products. To address the importance of TGB functions in vascular transport, we used an experimental system based on grafted plants and trans-complementation, to define co-translocated viral products and the minimal requirements for viral exit from the plant vasculature. Evidence is presented that TGBp1 is co-translocated with viral RNA and CP and that, once viral RNA is loaded into the phloem translocation stream, it can exit in sink tissues and replicate in the absence of TGBp2-3. These results are discussed in the context of the recent finding that TGBp1 can mediate the suppression of signaling involved in systemic gene silencing
Sequences in the 5′ Leader of Mason-Pfizer Monkey Virus Which Affect Viral Particle Production and Genomic RNA Packaging: Development of MPMV Packaging Cell Lines

AbstractWe used a series of deletion mutations in the 5′ untranslated region of the prototype D type retrovirus, Mason-Pfizer Monkey Virus (MPMV), to analyse RNA encapsidation. A region was identified upstream of the major splice donor which reduced particle production but had a proportionally greater effect on RNA packaging. A small deletion downstream of the splice donor had little effect on RNA production and caused no significant packaging defect. A large deletion encompassing the end of the primer binding site down to the splice donor had a dramatic effect, disrupting viral protein synthesis. Stable cell lines were produced containing packaging-defective virus. These first-generation packaging cell lines were used to package and transfer an MPMV-based vector
Cleavage of Vesicular Stomatitis Virus Matrix Protein Prevents Self-Association and Leads to Crystallization

AbstractThe matrix protein (M) of vesicular stomatitis virus is responsible for the budding of newly formed virions out of host cells. In vitro, it has been shown to self-associate, a property that may be related to the role of M in virus assembly but also prevents crystallization. Using limited proteolysis by thermolysin, we have isolated and characterized two soluble fragments of the protein that remain noncovalently associated. The digestion product does not self-associate nor is it recruited in aggregates formed by intact M molecules. These results identify a peptide, located at the surface of the protein and disorganized by thermolysin cleavage, responsible for M self-association. The thermolysin-resistant core of M has been crystallized and the crystals diffract to 2-Å resolution
Enzymatic Depalmitoylation of Viral Glycoproteins with Acyl-Protein Thioesterase 1 in Vitro

AbstractMany glycoproteins of enveloped viruses as well as cellular proteins are covalently modified with fatty acids. Palmitoylation is often reversible, but the enzymology of this hydrophobic protein modification is not understood. Recently a cytosolic enzyme designated acyl-protein thioesterase 1 (APT1) was purified, which depalmitoylates several cellular proteins. Since hitherto no transmembrane proteins have been tested as substrates for APT1 we have investigated whether palmitoylated viral membrane glycoproteins can be deacylated by use of this enzyme. Recombinant APT1 was purified from Escherichia coli, and depalmitoylation of [3H]palmitate-labeled glycoproteins present in virus particles was measured by SDS–PAGE, fluorography, and scanning densitometry. We find that APT1 causes rapid and almost complete cleavage of fatty acids from the G-protein of vesicular stomatitis virus, hemagglutinin proteins of influenza A and C virus, and E2 of Semliki Forest virus (SFV). In contrast, E1 of SFV is largely resistant against APT1 activity. This substrate specificity of APT1 was also observed using microsomes prepared from SFV-infected cells. Our data emphasize the potential of APT1 as a tool for functional analysis of protein-bound fatty acids
Involvement of Cellular Cytoskeleton Components in Antibody-Induced Internalization of Viral Glycoproteins in Pseudorabies Virus-Infected Monocytes

AbstractAddition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane-anchored viral glycoproteins and this may interfere with antibody-dependent cell lysis. We investigated the role of actin, microtubules, clathrin, and dynein, the major cellular components involved in physiological endocytosis during this virological internalization. Porcine monocytes were infected in vitro for 13 h and afterward treated with different concentrations of colchicine, cytochalasin D, latrunculin B, and amantadine-HCl, which inhibit polymerization of microtubules, actin/clathrin, actin, and clathrin, respectively. This resulted in a significant reduction of internalization compared to the nontreated control, indicating that these components are involved in the process. A double labeling was performed during the internalization process and a clear colocalization of actin, microtubules, clathrin, and dynein with the viral glycoproteins was observed at different stages during the internalization process. We conclude that these cellular components are used by PrV to generate the antibody-induced internalization of viral glycoproteins
The bet Gene of Feline Foamy Virus Is Required for Virus Replication

AbstractFoamy viruses (FV) are complex retroviruses with additional bel genes located between env and the 3′ long-terminal repeat. The functions of the bel 2 and bet genes are unknown and both are dispensable for replication of the prototypic human foamy virus in cell cultures. We examined the function(s) of bel 2 and bet of the distantly related feline foamy virus (FFV) in the proviral context. Mutagenesis was used to alter the Bel 2 and Bet or to abrogate their expression. The Bel 2/Bet mutants showed a 1000-fold reduced viral titer in feline kidney cells; in human 293T cells, viral titer was only about 10-fold reduced compared to wild-type FFV. In both cell types, the Bel 2/Bet mutations resulted in a reduced release of FFV particles. The results indicate that FFV Bet is required for efficient virus replication. The functions of the Bel 2 and Bet proteins are discussed
Differences in DNA Binding Specificity among Roseolovirus Origin Binding Proteins

AbstractThe Roseolovirus genus of the Betaherpesvirinae consists of the very closely related viruses, human herpesvirus 6 variants A and B (HHV-6A and HHV-6B) plus the somewhat more distantly related human herpesvirus 7 (HHV-7). The roseoloviruses each encode a homolog of the alphaherpesvirus origin binding protein (OBP) which is required for lytic DNA replication. In contrast, members of the other betaherpesvirus genera, the cytomegaloviruses, initiate DNA replication by a different mechanism. To better understand the basis of roseolovirus OBP sequence specificity, we investigated their ability to recognize each other's binding sites. HHV-6A OBP (OBPH6A) and HHV-6B OBP (OBPH6B) each bind to both of the HHV-7 OBP sites (OBP-1 and OBP-2) with similar strengths, which are also similar to their nearly equivalent interactions with their own sites. In contrast, HHV-7 OBP (OBPH7) had a gradient of binding preferences: HHV-7 OBP-2 > HHV-6 OBP-2 > HHV-7 OBP-1 > HHV-6 OBP-1. Thus, the roseolovirus OBPs are not equally reciprocal in their recognition of each other's OBP sites, suggesting that the sequence requirements for the interaction of OBPH7 at the OBP sites in its cognate oriLyt differ from those of OBPH6A and OBPH6B
Effect of Influenza Virus Matrix Protein and Viral RNA on Ribonucleoprotein Formation and Nuclear Export

AbstractThe formation of influenza virus ribonucleoprotein (RNP) is a necessary step in viral assembly and maturation in infected cells, but the mechanism remains incompletely understood. Influenza virus proteins such as matrix (M1) and cellular proteins have been implicated in assembly and transport of RNP. To study the assembly of RNP and the translocation of RNP complexes in cells, RNPs were reconstituted from nucleoprotein (NP), M1, and viral RNA (vRNA) synthesized in vitro. The syntheses were accomplished using specific plasmids in a system coupling transcription and translation under the control of the T7 promoter. The density of the resulting RNP complexes was analyzed by glycerol gradient centrifugation and the morphology was examined by transmission electron microscopy. Protomers of NP self-assembled into circular oligomers regardless of the presence of vRNA or M1. However, helical structures similar in conformation and density to RNPs purified directly from influenza virus were formed only when M1 and vRNA were also present. In the absence of vRNA, no helical structures were formed from NP and M1. The plasmids also contained the CMV promoter, which permitted expression of M1, NP, and vRNA in Madin–Darby canine kidney (MDCK). M1 and NP were both present in the cytoplasm of MDCK also expressing vRNA, but NP was retained in the nucleus of cells expressing M1 without vRNA. Our data demonstrate for the first time that vRNA and M1 together promote the self-assembly of influenza virus NP into the quaternary helical structure typical of the viral RNP. The results also indicate that the interaction of NP with vRNA and M1 in a system devoid of other viral proteins can lead to translocation of RNP from nucleus to cytoplasm
Sendai Virus Wild-Type and Mutant C Proteins Show a Direct Correlation between L Polymerase Binding and Inhibition of Viral RNA Synthesis

AbstractThe Sendai virus C proteins, C′, C, Y1, and Y2, are a nested set of four independently initiated carboxy-coterminal proteins encoded on the P mRNA from an alternate reading frame. Together the C proteins have been shown to inhibit viral transcription and replication in vivo and in vitro and C′ binds the Sendai virus L protein, the presumed catalytic subunit of the viral RNA polymerase. To identify amino acids within the C′ protein that are important for binding L, site-directed mutagenesis of the gstC′ gene was used to change conserved charged amino acids to alanine, generating nine mutants. Additionally, a tenth natural mutant, gstF170S, was also constructed. Six of the gstC′ mutants, primarily in the C-terminal half of C′, exhibited a defect in the ability to bind L protein. The mutants were assayed for their effect on in vitro transcription and replication from the antigenomic promoter, and the data suggest in all but one case a direct correlation between the ability of C to bind L and to inhibit these steps in RNA synthesis. Further studies with two nonfusion C mutants showed that this correlation was specifically due to the C′ portion, and not the gst portion, of the fusion proteins. To study their individual functions, each of the four C proteins was fused downstream of glutathione S-transferase. The gstC′, gstC, gstY1, and gstY1 fusion proteins were all able to bind L protein and to inhibit viral mRNA and (+)-leader RNA synthesis, and antigenome replication in vitro. In addition, the nonfusion C, Y1, and Y2 proteins all inhibited transcription. The inhibition of (+)-leader RNA and mRNA synthesis by wt C proteins (nonfusion) showed nearly identical dose-response curves, suggesting that inhibition occurs early in RNA synthesis
Low-Level Expression of Functional Foamy Virus Receptor on Hematopoietic Progenitor Cells

AbstractFoamy viruses have several qualities favorable for vector development: they are not known to cause disease; they can transduce stationary cells; and the foamy virus receptor is expressed on a wide variety of cells. Here, we analyzed the level of virus receptor expression on hematopoietic progenitor cells. Foamy virus binding was measured by a flow cytometric assay and was found to be considerably reduced in hematopoietic progenitors cell lines as well as in primary CD34+ cells when compared to fibroblasts. Retroviral vectors based on murine leukemia virus (MLV) pseudotyped with a foamy virus envelope transduced hematopoietic cell lines with a more than 10-fold lower efficiency than fibroblasts. Moreover, less than 1% of primary CD34+ hematopoietic progenitor cells were transduced with the foamy virus pseudotypes, while gene transfer efficiencies of 8–40% were achieved using pseudotypes with amphotropic envelope or the G protein of vesicular stomatitis virus. In conclusion, the expression of functional foamy virus receptors on hematopoietic progenitors cells was found to be insufficient to achieve high levels of gene transfer into CD34+ hematopoietic progenitor cells with cell-free vector supernatants using current transduction protocols
Effect of Transforming Growth Factor-β1 on the Cell Growth and Epstein–Barr Virus Reactivation in EBV-Infected Epithelial Cell Lines

AbstractTransforming growth factor (TGF)-β1 is a multifunctional cytokine that plays important roles in regulating cell growth and differentiation in many biological systems. In this study, we found that gastric tissue-derived Epstein–Barr virus (EBV)-infected epithelial cell lines GT38 and GT39 had resistance to TGF-β1-mediated growth inhibition and apoptosis compared to a TGF-β1-susceptible gastric carcinoma cell line HSC-39. However, TGF-β1 partially induced EBV reactivation in GT38 and GT39 cells, as shown by the induction of EBV immediate-early BZLF1 RNA and its protein product ZEBRA and early antigen-D. The expressions of TGF-β receptor I and II were detected in GT38 and GT39 cells by Northern and Western blot analyses. Both cell lines spontaneously produced the TGF-β1, which was sufficient for inhibiting cell growth of HSC-39 cells. Taken together, these data suggest that TGF-β1 may be a key factor for EBV reactivation and selective growth of EBV-infected epithelial cells in vivo
Growth Transformation of Primary Epithelial Cells with a NPC-Derived Epstein–Barr Virus Strain

AbstractThe Epstein–Barr virus (EBV) is associated with two major human epithelial malignancies, where it is likely to play a role in the malignant phenotype: undifferentiated nasopharyngeal carcinoma (100% of cases) and gastric carcinomas (about 10% of cases). We and others have obtained growth transformation of monkey kidney primary epithelial cells by transfection of viral DNA, especially with the BARF1 gene of EBV (Wei et al., 1997). We now report that the same type of primary epithelial cells can be growth-transformed using EBV particles derived from a nasopharyngeal carcinoma tumor line. Not only can these EBV-infected cells grow over 100 passages, escaping senescence, in contrast to their noninfected counterparts, but they can also survive and proliferate at very low cell density. Several subclones were characterized in terms of viral gene expression. All these clones gave a similar pattern, with detection of EBNA1 and BARF1 proteins but absence of LMP1. CD21, which is the main EBV receptor on B lymphocytes, was not expressed on parental monkey kidney epithelial cells nor on EBV-infected cell clones. This model of epithelial cell transformation will be useful for a better investigation of EBV functions critical for oncogenesis of epithelial cells
Generation of a High-Capacity Hybrid Vector: Packaging of Recombinant Adenoassociated Virus Replicative Intermediates in Adenovirus Capsids Overcomes the Limited Cloning Capacity of Adenoassociated Virus Vectors

AbstractGene therapy aims to complement or, ideally, correct defective genes. The broad clinical application of this emerging technology requires the development of safe high-capacity gene delivery vehicles that combine efficient transduction of dividing as well as quiescent cells with sustained transgene expression. Here we present a new hybrid vector system that unites favorable attributes of adenoassociated virus (AAV) and adenovirus (Ad) vectors in a single particle. This was achieved by inclusion of Ad packaging elements in different sized recombinant AAV genomes. In the presence of AAV replicative functions and a recombinant helper Ad, AAV/Ad hybrid particles were generated via encapsidation of AAV-dependent replicative intermediates into Ad capsids. In stringent in vitro models based on transduction of proliferating cells we show that AAV/Ad hybrid vectors are superior to Ad vectors in establishing prolonged transgene expression and can be used to deliver DNA fragments of at least 27 kb
Construction of Chimeric Arteriviruses Reveals That the Ectodomain of the Major Glycoprotein Is Not the Main Determinant of Equine Arteritis Virus Tropism in Cell Culture

AbstractThe recent development of arterivirus full-length cDNA clones makes possible the construction of chimeric arteriviruses for fundamental and applied studies. Using an equine arteritis virus (EAV) infectious cDNA clone, we have engineered chimeras in which the ectodomains of the two major envelope proteins, the glycoprotein GP5 and the membrane protein M, were replaced by sequences from envelope proteins of related and unrelated RNA viruses. Using immunofluorescence microscopy, we monitored the transport of the hybrid GP5 and M proteins to the Golgi complex, which depends on their heterodimerization and is a prerequisite for virus assembly. The only viable chimeras were those containing the GP5 ectodomain from the porcine (PRRSV) or mouse (LDV) arteriviruses, which are both considerably smaller than the corresponding sequence of EAV. Although the two viable GP5 chimeras were attenuated, they were still able to infect baby hamster kidney (BHK-21) and rabbit kidney (RK-13) cells. These cells can be infected by EAV, but not by either PRRSV or LDV. This implies that the ectodomain of the major glycoprotein GP5, which has been postulated to be involved in receptor recognition, is not the main determinant of EAV tropism in cell culture