pinecone/core-2020-05-10-deduplication huggingface dataset:
The Native Structure of the Human Immunodeficiency Virus Type 1 RNA Genome Is Required for the First Strand Transfer of Reverse Transcription

AbstractRetroviral particles contain two genomic RNAs of ∼9 kb that are linked in a noncovalent manner.In vitrostudies with purified transcripts have identified particular RNA motifs that contribute to the RNA-dimerization reaction, but the situation may be more complex within virion particles. In this study, we tested whether the primer-binding site (PBS) of the human immunodeficiency virus type 1 (HIV-1) RNA genome and the associated tRNALys3primer play a role in the process of RNA dimerization. Deletion of the PBS motif did not preclude the formation of RNA dimers within virus particles, indicating that this motif and the tRNA primer do not participate in the interactions that control RNA packaging and dimerization. Genome dimerization has been proposed to play a role in particular steps of the reverse transcription mechanism. To test this, reverse transcription was performed with the native RNA dimer and the heat-denatured template. These two template forms yielded equivalent levels of minus-strand strong-stop cDNA product, which is an early intermediate of reverse transcription. However, melting of the RNA dimer precluded the next step of reverse transcription, in which the minus-strand strong-stop cDNA is translocated from the 5′ repeat element to the 3′ repeat element. The results suggest that the conformation of the dimeric RNA genome facilitates the first strand-transfer reaction of the reverse transcription mechanism
Combined DNA Immunization with the Glycoprotein Gene of Viral Hemorrhagic Septicemia Virus and Infectious Hematopoietic Necrosis Virus Induces Double-Specific Protective Immunity and Nonspecific Response in Rainbow Trout

AbstractGlycoprotein (G) of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) contains several neutralizing epitopes. However, recombinant G protein never matches intact viral particles for immunogenicity. DNA immunization offers the possibility to deliver the antigen through the cellular machinery, thus mimicking natural infection. We constructed pCDNA gVHS and pCDNA gIHN plasmids with the G gene of VHSV and IHNV under the control of the CMV promoter, and we tested the plasmids for the accurate G protein expression prior to their use in fish immunization. Following intramuscular injection to adult rainbow trout, plasmid DNA was found inside the muscle cells shortly after injection and was still present 45 days later. mRNA of the G protein was detected in muscle tissue extracts, and the G protein was found within muscle cells at the site of injection. This resulted in the synthesis of high levels of specific neutralizing and protective antibodies. Fish injected with pCDNA gVHS and pCDNA gIHN in combination responded similarly to fish receiving one recombinant plasmid. In addition to the elicitation of a strong humoral response, DNA immunization was able to activate specialized cells of the immune system as well as nonspecific defense mechanisms, since mRNAs of MHC class II and Mx were strongly activated at the site of injection
The Role of v-Fgr Myristoylation and the Gag Domain in Membrane Binding and Cellular Transformation

AbstractThev-fgroncogene encodes a chimeric oncoprotein composed of feline sarcoma virus (FeSV)-derived gag and cellular-derived actin and c-Fgr sequences. v-Fgr is myristoylated and membrane bound, two criteria which must be met forsrckinases to induce cellular transformation. Although inhibition of myristoylation resulted in a decreased ability of v-Fgr to sediment with membranes from an NIH-3T3 P100 fraction, deletion of the gag domain caused nearly all of the protein to remain unbound and cytosolic. Systematic deletions within gag indicate that while amino acids 3 through 9 are critical determinants of myristoylation and/or define a domain which directs membrane localization, these residues cooperate with additional gag sequences when anchoring the protein to the plasma membrane. Furthermore, nonmyristoylated and/or cytoplasmic variants of v-Fgr failed to induce anchorage-independent growth of NIH-3T3 cells, indicating that proper subcellular localization of v-Fgr is a key factor in its ability to induce transformation
Psittacine Beak and Feather Disease Virus Nucleotide Sequence Analysis and Its Relationship to Porcine Circovirus, Plant Circoviruses, and Chicken Anaemia Virus

AbstractCloning and sequencing of the circular, single-stranded DNA of one isolate of psittacine beak and feather disease virus (BFDV) demonstrate a genome composed of a circular molecule of 1993 nucleotide bases. An analysis of the assembled replicative form demonstrated seven open reading frames (ORFs) (three in the virion strand and four in the complementary strand), potentially encoding seven viral proteins of >8.7 kDa. High amino acid sequence similarity was demonstrated between a potential 33.3-kDa protein product of ORF1 of BFDV and the replicase-associated protein of porcine circovirus (PCV), subterranean clover stunt virus, and faba bean necrotic yellows virus. However, significant similarity in nucleotide or amino acid sequences was not present between BFDV and chicken anaemia virus. A potential stem–loop structure similar to that found in PCV and plant circoviruses was present in the putative encapsidated strand of the BFDV genome. At the top of this structure, a nonanucleotide motif (TAGTATTAC) similar to that of PCV, plant circoviruses, and geminiviruses also was recognised. Comparison of the deduced amino acid sequences of ORF2 of BFDV and PCV demonstrated 29.1% identity, and in both viruses, ORF2 is located on the complementary strand, beginning close to or within the hairpin stem. Our findings provide further evidence of a close relationship among BFDV, PCV, and plant circoviruses but not chicken anaemia virus
Macaques Infected with Cloned Simian Immunodeficiency Virus Show RecurringnefGene Alterations

AbstractWe comparednefgene sequences isolated by polymerase chain reaction from peripheral blood lymphocyte DNA of macaques that had been inoculated with either biologically (E11S) or molecularly (clone 8) cloned SIV/Mne. Two samples from each animal obtained either early (weeks 2–8) or late (weeks 21–137) after infection were analyzed. Three substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and two other substitutions were seen in all except one. Two of the common exchanges are located ∼40 residues apart in the Nef core sequence but are juxtaposed on the tertiary structure as judged by computer modeling using the structure of the HIV Nef core protein as a guide. Most recurrentin vivochanges replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIV/Sm clade. Recombinant virus containing a macaque-adapted (MAnef)nefon the clone 8 backbone was 3-fold more infectious on SMAGI cells than the original virus. A lymphocyte line infected with SIV-clone 8-MAnefcontained a large proportion of cells carrying provirus with defectivenefgenes. These findings suggest that thenefgene of the cloned SIV/Mne had undergone attenuating mutations during propagation in tissue culture that were “corrected”in vivo
Aberrant Expression of a Cytokeratin in a Subset of Hepatocytes during Chronic WHV Infection

AbstractChronic infection of woodchucks with woodchuck hepatitis virus (WHV) invariably leads, within 2–4 years, to the appearance of hepatocellular carcinoma (HCC). HCC is preceded by an extended period of chronic liver damage, probably resulting from the immune response to viral antigens. It may be that infection itself also induces changes in the hepatocyte population. To begin to identify some of the changes in the liver prior to the appearance of HCC, monoclonal antibodies (MAbs) were generated from mice immunized with hepatocytes from a woodchuck chronically infected with WHV or with a tumor lysate. Immunofluorescence microscopy was used to select MAbs that reacted with host markers whose patterns of expression would distinguish chronically infected from uninfected liver or from liver tumors. One of these MAbs (2F2) reacted strongly with a subset of hepatocytes in chronically infected liver; a similar staining pattern was not detected in uninfected or transiently infected liver. Evidence is presented that this strong staining reaction reflects the overexpression or accumulation of the hepatocyte-specific intermediate filament protein, cytokeratin K18, a protein previously implicated in cryptogenic cirrhosis of the liver in humans (Ku, N. O., Wright, T. L., Terrault, N. A., Gish, R., and Omary, M. B.J. Clin. Invest.99: 19–23, 1997). Double immunofluorescent staining with antibodies to K18 and M-envelope protein of WHV suggested that strong reactivity to K18 was limited to cells expressing high levels of one or both of the large viral-envelope proteins, M and L; however, high expression of these viral proteins was not always associated with a strong K18 staining reaction
Regulated Lentiviral Packaging Cell Line Devoid of Most Viralcis-Acting Sequences

AbstractPackaging cell lines derived from human immunodeficiency virus-1 (HIV-1) are promising tools forin vivosomatic cell gene therapy protocols due to the ability of lentiviruses to infect nondividing cells. We describe here the generation of a safe, stable HIV-1 packaging cell line capable of expressing all of the HIV-1 structural, enzymatic, and regulatory proteins but lacking majority of thecis-acting sequences. The use of an inducible expression system circumvented the cytotoxic and cytostatic effects associated with the expression of some of the HIV-1 viral proteins. Reverse transcriptase activity was detectable in the supernatant from the stable packaging line 1 day after induction, while vector titers peaked 5 days postinduction. Vector titers of up to 3.5 × 104infectious units/ml (IU/ml) were maintained through 8 months following the establishment of the cell line. Lineage-specific gene delivery can be achieved from this established cell line as viral stocks obtained specifically infect CD4+target cells. Moreover, this cell line provides a safe and easy to use system for screening of drugs that inhibit HIV-1 replication
Interactions of Marburg Virus Nucleocapsid Proteins

AbstractIn this study, the components of Marburg virus nucleocapsid complex were determined, and interactions between the compounds were investigated. Using salt dissociation of isolated virions, four proteins (NP, VP35, VP30, and L) remained attached to the core complex. Same proteins were detected intracellularly to be localized in MBGV-induced inclusion bodies, which are presumed to represent areas of nucleocapsid formation. To investigate interactions between the four proteins, immunofluorescence analysis of coexpressed proteins was carried out. Complexes between NP-VP35 and NP-VP30 were formed, which was demonstrated by redistribution of VP35 and VP30 into NP-induced inclusion bodies. Furthermore, complexes between L and VP35 were detected by coimmunoprecipitation. Using deletion mutants of L, the binding site of VP35 on L could be restricted to the N-terminal 530 amino-acid residues. Coexpression of NP, VP35, and L led to the formation of a triple complex where VP35 linked NP and L. The detected complexes are presumed to represent the key components of the MBGV transcription and replication machinery
A Classical Bipartite Nuclear Localization Signal on Thogoto and Influenza A Virus Nucleoproteins

AbstractWe have previously shown that the nucleoprotein (NP) of Thogoto virus (THOV), a tick-borne member of the Orthomyxoviridae family, accumulates in the cell nucleus. Here we demonstrate that THOV NP contains a motif (KRxxxxxxxxxKTKK) at amino acid positions 179–193 that represents a classical bipartite nuclear localization signal (NLS). This sequence motif (named cNLS) was able to translocate a cytoplasmic 80-kDa reporter protein into the nucleus. Targeted mutations substituting lysines for alanines in the downstream cluster of the bipartite motif abolished the capacity of cNLS to mediate nuclear import. In contrast, identical mutations had no effect on nuclear localization when introduced into THOV NP, indicating that additional transport signals are present in NP. Amino-acid sequence comparisons revealed that THOV NP lacks the N-terminal nonconvential NLS (named here nNLS), which has been implicated in nuclear import of influenza A virus NP. Accordingly, THOV NP failed to interact in coprecipitation assays with the cellular NPI-1/3 transport factors of the karyopherin α family. A highly conserved motif identified in THOV NP was the so-called nuclear accumulation sequence (NAS). Mutating NAS alone, or in combination with cNLS, had no gross effect on the intracellular distribution of the protein, indicating that a functional NAS is not required for nuclear accumulation of THOV NP in mammalian cells. We also studied nuclear transport of influenza A/PR/8/34 virus NP. Interestingly, we found a cNLS motif at amino acid positions 198–216 in addition to the previously described nonconventional nNLS. To further assess the functional role of cNLS, nNLS, and NAS, we analyzed single, double, and triple mutants of influenza A virus NP. When nNLS was destroyed, the protein stayed in the cytoplasm as expected. When NAS was disrupted in addition to nNLS, the double mutant accumulated in the nucleus, suggesting that cNLS was active. Indeed, when cNLS was also inactivated, the triple mutant protein localized again predominantly to the cytoplasm. These findings suggest that NP of orthomyxoviruses have two independent NLSs, namely cNLS and nNLS. They further suggest that NAS and NLSs may assume opposing roles in nucleocytoplasmic transport of NP
Transfectant Influenza A Viruses Are Effective Recombinant Immunogens in the Treatment of Experimental Cancer

AbstractUsing reverse genetics methods, we constructed three different transfectant influenza A viruses encoding an Ld-restricted, nine amino-acid-long fragment, corresponding to amino-acid residues 876–884, of β-galactosidase (β-gal). Sequences encoding this epitope were nested within the hemagglutinin (HA) or neuraminidase (NA) open reading frames. Alternatively, an independent β-gal mini-gene, preceded by an endoplasmic reticulum insertion signal sequence, was placed in a bicistronic arrangement in the NA RNA segment of the virus. All three transfectants mediated the presentation of the epitope to a β-gal-specific CTL clone. Furthermore, each of the three transfectant viruses expressing the β-gal fragment elicited specific cytolytic responsesin vivo.Most importantly, these H1N1 transfectants mediated the regression of established murine pulmonary metastases. Tumor regression in mice was also achieved in the presence of preexisting immunity against an H3N2 influenza A virus serotype. Nononcogenic and nonintegrating, transfectant influenza A viruses are attractive candidates for development as antitumor vaccines
Targeted Integration of Adeno-Associated Virus-Derived Plasmids in Transfected Human Cells

AbstractAdeno-associated virus (AAV) integrates its genomic DNA into a defined region of human chromosome 19 (AAVS1). The specificity of integration is dependent on the presence of the inverted terminal repeats (ITR) and on expression of therepgene. To develop vectors capable of targeting the insertion of a selected DNA sequence into a specific location of human chromosome, we determined whether therepgene can mediate site-specific integration when cloned outside of an ITR-flanked transgene cassette. HeLa and Huh-7 cells were transfected with a plasmid containing therepgene, as well as the green fluorescent protein (GFP) and neomycin (neo) resistance gene inserted between the ITRs of AAV. Southern blot analysis of individual clones detected Rep-mediated site-specific integration of the ITR-flanked DNA in 25% and 12% of the HeLa and Huh-7 clones, respectively. The localization of the GFP-Neo sequence on chromosome 19 also was confirmed by fluorescentin situhybridization analysis of the transfected HeLa clones. Sequence analysis of the ITR–AAVS1 junction of one of the transfected Huh-7 clones indicated that the insertion of the ITR DNA fragment had occurred at nucleotide 1003. These results have implications for the development of AAV-derived vectors capable of directing the site-specific integration of a gene of interest
Transgenically Expressed Cucumber Mosaic Virus RNA 1 Simultaneously Complements Replication of Cucumber Mosaic Virus RNAs 2 and 3 and Confers Resistance to Systemic Infection

AbstractTobacco plants transformed with a cDNA copy of RNA 1 of the Fny strain of cucumber mosaic virus (CMV) promoted the asymptomatic accumulation of inoculated viral RNAs 2 and 3, which could be detected in noninoculated leaves, suggesting that the transgene also permitted viral long-distance movement. Typical symptoms of infection appeared later and correlated with the appearance of viral RNA 1 regenerated from the transgenic mRNA. Although all R0-generation plants were susceptible to Fny-CMV, one line displaying variable susceptibility to the virus in R1-and R2-generations led to selected R3-generation lines with systemic resistance to Fny-CMV. In the inoculated leaves of resistant plants, a dramatic decrease in the accumulation of viral RNA 1 was observed, relative to susceptible plants. No viral RNAs were detected in noninoculated leaves of the resistant plants, but such leaves were susceptible to infection. Furthermore, these leaves could sustain replication of inoculated CMV RNAs 2 and 3, indicating that a complete transgene-silencing had not been induced. Although a transgene-mediated, CMV RNA 1-suppression occurred in the inoculated leaf of resistant plants, the absence of a complete systemically acquired silencing suggests the existence of additional interferences with viral infection that prevented systemic infection by viral RNAs 2 and 3
AcMNPV Late Expression Factor-5 Interacts with Itself and Contains a Zinc Ribbon Domain That Is Required for Maximal Late Transcription Activity and Is Homologous to Elongation Factor TFIIS

AbstractThe late expression factor-5 gene (lef-5) ofAutographa californicamultinucleocapsid polyhedrovirus (AcMNPV) is required for late gene expression. In this paper, we demonstrate that LEF-5 interacts with itself in the yeast two-hybrid system and in glutathione–S-transferase affinity assays. Deletion analysis suggested that the C-terminal 71 amino acids (aa) were not required for interaction. However, all deletions tested involving the N-terminal 194 aa significantly reduced LEF-5:LEF-5 interaction. LEF-5 or LEF-5 deletion mutants were transfected into Sf-9 cells with the full complement of genes required for baculovirus late transcription. All deletion clones tested reduced expression of a β-glucuronidase (GUS) reporter gene under control of the latevp39capsid promoter. Amino-acid sequence analysis of LEF-5 identified a previously unreported domain within the C-terminal 32 aa that is homologous to the zinc ribbon domain of RNA polymerase II elongation factor IIS (TFIIS) from a variety of taxa. Molecular modeling of the putative LEF-5 Zn ribbon using the NMR data available for the Zn ribbon of TFIIS suggested that this domain could fold into a Zn ribbon structure similar to TFIIS. Alanine scanning mutagenesis of amino acids predicted to be important for functioning of the LEF-5 ribbon structure significantly reduced LEF-5 activity in transient expression assays. Mutations changing the amino acids predicted to coordinate Zn2+caused a reduction in activity similar to that when the domain was eliminated completely
Differences in the Evolutionary Pattern of Feline Panleukopenia Virus and Canine Parvovirus

AbstractCanine parvovirus (CPV) suddenly appeared in the late 1970s after which it showed continuous antigenic changes. Virological and molecular genetic analyses mainly focused on feline panleukopenia virus (FPLV) were conducted in this study because FPLV is the suspected ancestor of CPV; the way in which FPLV evolves may help to explain the emergence of CPV. Analysis of escape mutants against FPLV-specific monoclonal antibody showed that viruses possessing CPV-like properties were not easily detected in FPLV virus stocks. Phylogenetic analysis revealed that the nonstructural protein 1 (NS1) and capsid protein 2 (VP2) genes of FPLV changed with time. A similar tendency, however, was not observed in the FPLV VP2 proteins. In contrast, the topology of the phylogenetic tree of VP2 proteins of CPV basically concurred with that of the VP2 genes. Analysis of the ratio of nonsynonymous and synonymous substitutions revealed that synonymous substitutions exceeded nonsynonymous substitutions in both the NS1 and VP2 genes of FPLV, even when the analysis focused on specific regions in the VP2 gene that are known to be located on the capsid surface. Comparison of the CPV VP2 genes revealed that nonsynonymous substitution was found to dominate over synonymous substitution in one specific region in the VP2 gene. These results suggested that FPLV has changed mainly by random genetic drift. In contrast, after the appearance of CPV, changes in the CPV VP2 gene appear to be partly selected by certain positive selection forces. CPV and FPLV are known to be closely related viruses genetically and biologically, but the evolutionary mechanisms of the two viruses appeared to be different
The C Terminus of E1A Regulates Tumor Progression and Epithelial Cell Differentiation

AbstractThe E1A gene of adenovirus has been considered both a dominant oncogene and a tumor suppressor. It has been reported to induce epithelial cell but to prevent myoblast differentiation. E1A enables oncogenes that are unable to transform primary cells on their own to do so, yet suppresses tumor progression toward invasion and metastasis. To try to reconcile the seemingly, conflicting E1A phenotypes, we examined the expression of epithelial cell specific and characterizing proteins in immortalized or tumorigenically transformed primary epithelial cells expressing wild-type E1A or a C-terminal mutant that has lost tumor suppressive abilities. All the cell types continued to express cytokeratin. Epithelial cell morphology, social behavior, and growth characteristics were retained by cells expressing wild-type E1A, even in the presence of an activated ras oncogene. Mutant E1A-expressing cells were less well differentiated even in the absence of ras. They were specifically defective in cell–cell junctional complexes, such as tight and adherens junctions and desmosomes. There was also a preference for those actin structures prominent in fibroblasts: stress fibers and filopodia, while in the wild-type E1A expressing cells, cortical actin and circumferential actin filaments were dominant. Thus the E1A-mutant-expressing cells were already predisposed to a more advanced tumor stage even when they were only immortalized and not transformed. The results suggest the possibility that the C terminus of E1A may be involved in regulating epithelial mesenchymal transitions, which have previously been linked to tumor progression