pinecone/core-2020-05-10-deduplication huggingface dataset:
Dose-Dependent Transduction of Vesicular Stomatitis Virus G Protein-Pseudotyped Retrovirus Vector into Human Solid Tumor Cell Lines and Murine Fibroblasts

AbstractWe examined the transduction efficiency of a VSV-G (vesicular stomatitis virus G protein)-pseudotyped vector encoding β-galactosidase (lacZ) into human solid tumor cell lines and murine fibroblasts, compared with that of an amphotropic vector carrying the same RNA sequence. The ratio of cells transduced with the VSV-G-pseudotyped vector corresponded closely to 1 − e−m.o.i., as predicted from a Poisson distribution of transduction to the entire cellular population, while this was not the case for the amphotropic vector. Here m.o.i. (multiplicity of infection) is defined as the ratio of input infectious units (titrated on the corresponding cell line) to the number of cells used for the transduction. At high m.o.i.s (values greater than 3), the VSV-G-pseudotyped vector transduced approximately 95% of the culture population of all cell lines examined. The transduction efficiency of the amphotropic vector, however, was not dose-dependent and reached a plateau or even decreased, especially at high m.o.i.; this may be attributable at least in part to the presence of envelope protein and noninfectious particles that compete for the receptor of infectious amphotropic virus. The copy number of integrated vector proviral DNA and the expression level of lacZ increased almost linearly with the dose of the VSV-G-pseudotyed vector, which could readily achieve multiple transduction of more than 10 copies per cell and afforded about 100-fold more transgene product than could be achieved with the amphotropic vector. These features of both the VSV-G-pseudotyped vector and the amphotropic vector were essentially unaffected by purification using centrifugation. These properties of the vector should be highly advantageous for gene transfer into entire populations of human tumor cell lines at a designed dosage
Higher Selection Pressure from Antiretroviral Drugs in Vivo Results in Increased Evolutionary Distance in HIV-1 pol

AbstractWe investigated the effect of selection pressures on evolution of HIV-1 pol in 51 patients after switching to a new antiretroviral combination reverse transcriptase (RT) inhibitor therapy. Evolution of the protease (PR) and RT reading frames were analysed separately. Pairwise evolutionary distances (ED) were calculated between sequences from baseline and week 8 and between baseline and week 48 of protocol therapy. ED were calculated for all substitutions and for synonymous and nonsynonymous substitutions separately. At week 8 when HIV RNA reduction (selection pressure) was high, significantly more divergence in pol in both synonymous and nonsynonymous substitutions was found in patients with substantial RNA reduction (strong responders). Separate analyses of PR and RT revealed significantly greater ED in the RT (under selection pressure) of strong compared with nonresponders, whereas divergence between PR genes (not under selection pressure) did not differ in those two groups. Such differential evolution indicates that PR and RT were genetically unlinked and suggests recombination. The rapid increase of ED over the first 8 weeks was followed by only a minimal further rise by week 48, suggesting that selection of preexisting quasispecies accounted for the early changes. A disproportionally high number of synonymous substitutions accounted for the observed divergence and indicated that such genetic changes may not be completely silent
SVMPA, a Mutant of Sindbis Virus Resistant to Mycophenolic Acid and Ribavirin, Shows an Increased Sensitivity to Chick Interferon

AbstractSVMPA is a mutant of Sindbis virus, selected for its ability to replicate in mycophenolic acid (MPA)-treated mosquito cells. SVMPA has another phenotype: although able to replicate normally in primary cultures of chick embryo fibroblasts (CEF), its replication is restricted in secondary cultures prepared from aged primary CEF cultures. The mutations responsible for these phenotypes mapped to the region of the viral genome that codes for nsP1. We report here that SVMPA has yet another phenotype. Relative to our standard Sindbis virus (SVSTD) from which it was derived, SVMPA shows an increased sensitivity to chick interferon, both crude interferon prepared from virus-infected cells and recombinant interferon. Characterization of viral mutants obtained after site-directed mutagenesis indicated that the same mutations responsible for the host restriction of SVMPA in secondary cultures of CEF were also responsible for its increased sensitivity to chick interferon
Biochemical and Functional Properties of a Palindromic Sequence Motif within the Hepatitis B Virus Enhancer 1

AbstractThe hepatitis B virus (HBV) enhancer 1 is a transcriptional element that contributes to the liver-specific regulation of HBV gene expression. We previously identified a novel protein binding site within the enhancer that contains an 8-bp palindromic sequence motif. This motif partially overlaps the binding sites for nuclear factor 1 and hepatocyte nuclear factor 3β (HNF3β). Moreover, we demonstrated that this novel site is recognized by a protein or proteins, tentatively designated as palindrome-binding factor (PBF), that cooperatively interact with HNF3β. In the present work, we have further examined the biochemical and functional attributes of PBF. Protein–DNA interaction studies indicate that three thymidine residues located at the 3′-end of the palindromic sequence motif are important for maximal PBF-binding activity. When protein–DNA complexes were photocrosslinked by exposure to ultraviolet (UV) light, a prominent polypeptide with an apparent molecular mass of 50 kDa was found to associate with the PBF-binding site. Furthermore, transient transfection studies support the hypothesis that PBF contributes to enhancer 1 activity by a combinatorial mechanism that involves at least one other cis-acting sequence motif, the HNF3β-binding site
Distinct Export Pathway Utilized by the Hepatitis B Virus Posttranscriptional Regulatory Element

AbstractThe posttranscriptional regulatory element (PRE) of hepatitis B virus is an RNA element important for the export of viral mRNA from the nucleus to the cytoplasm. The cellular export pathway utilized by the PRE is controversial. We present data showing that PRE-dependent export is blocked by vesicular stomatitis virus matrix protein, an inhibitor of all cellular RNA export other than tRNA export. It is also blocked by a mutated form of Ran-binding protein 1, which blocks export mediated by the human immunodeficiency virus Rev and Rev-response element (RRE) but not export mediated by the simian retrovirus constitutive transport element (CTE). On the other hand, PRE-dependent export is not blocked by either TAgRex or leptomycin B, two agents that prevent Rev/RRE-mediated export. Therefore, PRE appears to utilize an export pathway different from that of Rev/RRE or CTE
HIV and SIV gp120 Binding Does Not Predict Coreceptor Function

AbstractInteraction of HIV and SIV Envelope (Env) proteins with viral coreceptors is a critical step in viral entry. By using a sensitive and specific gp120 binding assay, we have identified a discordance between the ability of a coreceptor to support Env-mediated membrane fusion and high-affinity binding of gp120. Direct binding of gp120 from the dual-tropic HIV-1 strain 89.6 was not detectable for any coreceptor that it uses for fusion, while detectable binding of gp120s from the R5 HIV-1 strains JRFL and CM235 and the SIV strain 239 was not measurable for many CCR5 chimeras and mutants that function efficiently as viral coreceptors. In comparison, binding of chemokines to these same mutants was highly predictive of their ability to signal. Thus, gp120 is more sensitive than chemokines to perturbations of CCR5 structure. We conclude that while chemokine binding to CCR5 is a good predictor of chemokine receptor function, gp120 binding does not always predict coreceptor function
Expression and Detection of Macrophage-Tropic HIV-1 gp120 in the Brain Using Conformation-Dependent Antibodies

AbstractHIV-1 envelope proteins gp120 and gp41 are likely to play a role in the pathogenesis of HIV-associated neurocognitive disorders. While detection of gp120 in HIV-infected cell cultures is easy, it has not yet been possible to identify gp120 in human or animal brains in situ. The difficulty in detecting gp120 could be due to low expression levels of the protein, to the shedding of gp120 from infected macrophages/microglia, or to the use of inappropriate gp-specific antibodies. We addressed these questions by analyzing the subcellular localization, oligomeric structure, and shedding behavior of gp120 from a macrophage-tropic, CCR5-dependent primary isolate, BX08, expressed by a Semliki Forest virus replicon (SFVenvBX08) in vitro. We used the same SFV system injected in vivo into the rat brain in an attempt to detect gp120 in situ. Our results show that gp120/41 is expressed as monomers, dimers, and trimers in cell culture. Immunocytochemical analysis revealed that intracytoplasmic gp120 can be recognized by an anti-V3 antibody, whereas gp120 at the plasma membrane is detected exclusively by a conformation-dependent antibody. In the rat brain, the SFV vector allows gene expression in neurons from day 3 to day 9 after injection without any apparent brain damage nor reactive astrogliosis. In SFVenvBX08-infected neurons only conformation-dependent antibodies allowed gp120 labeling. These results suggest that previous difficulties in detecting gp120 in brain tissues may be due to the use of antibodies which were unable to recognize gp120 at the plasma membrane
Symptom Attenuation by a Satellite RNA in Vivo Is Dependent on Reduced Levels of Virus Coat Protein

AbstractMany plant RNA viruses provide replication and encapsidation functions for one or more satellite RNAs (sat-RNAs) that can modulate the symptoms of the associated helper virus. Sat-RNA C, a virulent sat-RNA associated with turnip crinkle virus (TCV), normally intensifies symptoms but can attenuate symptoms if the TCV coat protein (CP) is replaced with that of cardamine chlorotic fleck carmovirus [Kong et al. (1995) Plant Cell 7, 1625–1634] or if TCV contains an alteration in the CP initiation codon (TCV-CPm) [Kong et al. (1997b) Plant Cell 9, 2051–2063]. To further elucidate the mechanism of symptom attenuation by sat-RNA C, the composition of the CP produced by TCV-CPm (CPCPm) was determined. Our results reveal that CPCPm likely has two additional amino acids at its N-terminus compared with wild-type TCV CP. TCV-CPm produces reduced levels of CP, and this reduction, not the two additional residues at the CP N-terminus, is responsible for symptom attenuation by sat-RNA C
Retrovirus DNA Termini Bound by Integrase Communicate in Trans for Full-Site Integration in Vitro

AbstractIntegration of linear retrovirus DNA involves the concerted insertion of the viral termini (full-site integration) into the host chromosome. We investigated the interactions that occur between long terminal repeat (LTR) termini bound by avian retrovirus integrase (IN) for full-site integration in vitro. Wild-type (wt) or mutant LTR donors that possess gain-of-function (“G”) or loss-of-function (“L”) for full-site integration activity were used. G LTR termini are characterized as having significantly higher strand transfer activity than the wt and the L LTR termini. L LTR mutations are classified as partially or extremely defective for strand transfer activity. The L mutations were further classified by their ability to either permit or block the assembly of G or wt LTR termini into nucleoprotein complexes capable of full-site strand transfer. We demonstrated that avian myeloblastosis virus IN bound to G LTR termini increased the incorporation of partially defective L LTR termini into nucleoprotein complexes that were capable of full-site integration. The observed full-site integration activity of these assembled nucleoprotein complexes appeared to be influenced by each individual IN–LTR complex in trans. In contrast, extremely defective L LTR termini exhibited the ability to effectively block the assembly of wt LTR termini into nucleoprotein complexes capable of full-site strand transfer. Data from nonspecific DNA competition experiments suggested that IN had an apparent higher affinity for G LTR donor termini than for partially defective L LTR donor termini as measured by full-site integration activity. However, assembled nucleoprotein complexes containing either two G or two L LTR donors were stable, having a similar half-life of ∼2 h on ice. The results suggest that LTR termini bound by IN exhibit an allosteric effect to modulate full-site integration in vitro. Similar regulatory controls also appear to exist in vivo between the wt U3 and wt U5 LTR termini in retroviruses as well as purified retrovirus preintegration complexes that promoted full-site integration in vitro
Defective nef Alleles in a Cohort of Hemophiliacs with Progressing and Nonprogressing HIV-1 Infection

AbstractDeletion of the nef gene results in viral attenuation and confers protection against challenge with wild-type simian immunodeficiency virus in macaques. Regarding HIV-1 infection, a few long-term nonprogressors (LTNP) with nef deletions have been described. In this study, the nef genes of a group of seven LTNP and eight progressors, all belonging to the same cohort of infected hemophiliacs, were analyzed by cloning and sequencing from both virion RNA and peripheral blood mononuclear cell-associated proviral DNA. Defective nef sequences coexisted with full-length nef open reading frames in five of seven LTNP and two of eight progressors. The proportion of disrupted nef sequences within each individual was significantly higher in LTNP (ranging from 10 to 63%) than in progressors (ranging from 9 to 21%) (P = 0.013). Moreover, in-frame small deletions predicting to encode Nef were found in all RNA- and DNA-derived clones from one LTNP and four progressors. A chimeric virus in which the nef gene of NL4.3 was substituted with the nef allele containing the deletion of two alanines at position 49–50 found in two progressors showed a defective replicative capacity compared to NL4.3 virus. In summary, hemophiliacs with either progressing or nonprogressing HIV-1 infection are characterized by the presence of defective nef variants
Comparison of the Assembly of the Bacteriophage T4 Clamp Loader Complex (gp44/62) Expressed in a cis versus trans Genomic Configuration

AbstractProper formation of the bacteriophage T4 DNA polymerase holoenzyme requires a wide spectrum of protein–protein and protein–DNA interactions among the DNA polymerase gp43, the sliding clamp gp45, and gp44/62, the clamp loader complex (CLC). The 44 and 62 proteins associate to form a tight complex maintained in a 4:1 ratio. The 44 and 62 genes are adjacent to each other on the T4 genome, are cotranscribed, and are translationally coupled. It has been suggested that translational coupling may play a role in the formation of the clamp loader complex and may control its stoichiometry. To examine the effect of coupling on the assembly of the complex, expression in trans of genes 44 and 62 was accomplished by cotransforming Escherichia coli with compatible, inducible plasmid vectors. A gp44/62 complex could be purified from such cells. The complex assembled in trans exhibited stoichiometry and ATPase activity identical to native complex. Burst sizes were determined to gauge the efficiency of clamp loader complex formation. When gp44 was supplied by a plasmid and gp62 was supplied by the T4 genome, complex formation was as efficient as in wild-type virus. However, when gp62 was supplied by plasmid and gp44 was supplied by the T4 genome, efficiency of complex formation was decreased. This decrease in the efficiency of complex formation was temperature dependent, being more pronounced at higher temperatures. At higher temperatures, a larger proportion of gp62 expressed from the plasmid was found to be present in an insoluble form. The decrease in efficiency of complex formation correlated to a decrease in solubility of the gene 62 protein
Modulation of Translational Efficiency by Contextual Nucleotides Flanking a Baculovirus Initiator AUG Codon

AbstractIn a previous study of translational regulation of a baculovirus gene, we observed that translation initiated at an unexpectedly high efficiency from an AUG codon found in what was believed to be a poor context (M.-J. Chang and G. W. Blissard, 1997, J. Virol. 71, 7448–7460). In the current study, we examined the roles of nucleotides flanking a baculovirus AUG initiator codon in modulating translation initiation in lepidopteran insect cells. The roles of nucleotides flanking the AcMNPV gp64 initiator codon were examined by site-directed mutagenesis and functional assays in transfected Sf9 cells. To eliminate potential cis-acting sequences and effects, the gp64 initiator context was cloned in-frame with a chloramphenicol acetyl transferase reporter gene and under the control of a heterologous promoter. All possible single-nucleotide substitutions were generated in positions −6 to −1 and +4 to +6, relative to the A of the initiator AUG codon, which was designated +1. Constructs were transfected into lepidopteran cells and translation products were quantified by an enzyme-linked immunosorbent assay procedure. Substitutions of pyrimidines or other nucleotides at the −3 position resulted in little or no detectable effect on translation efficiency. In contrast, specific substitutions at the +4 and +5 positions resulted in approximately 2- to 3-fold increases in translation. Substitution of A in the +4 position resulted in an approximately 3-fold increase in translation, and substitution of any nucleotide for T in the +5 position resulted in approximately 1.9- to 2.8-fold increases. Substitutions at other positions (−6 to −1 and +6) resulted in no detectable increase or decrease in translation efficiency. These experimental results suggest an optimal initiator context of 5′-N N N N N N A U G A a/c/g N-3′ for efficient translation initiation in lepidopteran cells. Consensus translation initiation contexts were generated from baculovirus genes and lepidopteran genes, then compared with the experimental results from the gp64 initiator context
Evidence for Two Nonoverlapping Functional Domains in the Potato Virus X 25K Movement Protein

AbstractTo study subdomain organization of the potato virus X (PVX) movement protein (MP) encoded by the first gene in the triple gene block (TGB), we mutated the 25-kDa TGBp1 protein. The N-terminal deletion of the helicase motifs I, IA, and II resulted in loss of the ATPase activity and RNA binding. A frameshift mutation truncating the C-terminal motifs V and VI gave rise to increase of the TGBp1 ATPase activity and had little effect on RNA binding in vitro. Fusions of the green fluorescent protein with 25-kDa MP and its derivative lacking motifs V–VI exhibited similar fluorescence patterns in epidermal cells of Nicotiana benthamiana leaves. Cell-to-cell movement of the 25K-deficient PVX genome was not complemented by the TGBp1 of Plantago asiatica mosaic potexvirus (PlAMV) but was efficiently complemented by a chimeric TGBp1 consisting of the N-terminal part of PlAMV protein (motifs I–IV) and the PVX-specific C-terminal part (motifs V–VI). These results suggest that NTP hydrolysis, RNA binding, and targeting to the specific cellular compartment(s) are associated with the N-terminal domain of the TGBp1 including the helicase motifs I–IV and that the C-terminal domain is involved in specific interactions with other virus proteins
The Evolution of Porcine Reproductive and Respiratory Syndrome Virus: Quasispecies and Emergence of a Virus Subpopulation during Infection of Pigs with VR-2332

AbstractGP5, the principal envelope glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV), contains a hypervariable region within the ectodomain which is responsible for generating diversity in field isolates. The purpose of this study was to gain insight into the possible origin of this diversity by following GP5 sequence changes in pigs exposed to PRRSV strain VR-2332 in utero. A region of the PRRS virus genome containing portions of ORF4 and ORF5 was amplified directly from tissues of infected pigs from birth to 132 days of age. We observed the emergence of a new PRRSV population, identified by a single nucleotide change in the ectodomain. The Asp to Asn change at amino acid 34 was also found as a minor component in pigs that expressed the wild-type sequence. The results from this study suggest that the variability in the ectodomain of ORF5 is the result of positive or negative selection, of which the mechanism remains to be determined
Posttranscriptional Regulation of US11 in Cells Infected with a Herpes Simplex Virus 1 Recombinant Lacking Both 222-bp Domains Containing S-Component Origins of DNA Synthesis

AbstractThe US11 gene of herpes simplex virus 1 maps in the unique sequences of the short component of the HSV-1(F) genome approximately 775 bp from the center of the DNA replication origin (OriS) and encodes a virion protein which binds RNA in sequence- and conformation-specific fashion, negatively regulates the accumulation of a prematurely terminated transcript of UL34, associates in the infected cell with the 60S ribosomal subunit, and, late in infection, accumulates in nucleoli. We report the following: (i) Deletion of a 222-bp sequence including OriS (ΔOriS) negatively affected the accumulation of the US11 protein without decreasing the accumulation of the US11 transcript. (ii) The defect, observed at all times after infection, was multiplicity independent, was unrelated to US11 protein stability, and apparently resulted from a cis-acting element since a coinfecting virus was unable to complement the ΔOriS virus. (iii) Transcription from the US11 promoter initiated from three sites on the ΔOriS virus. Transcripts initiated from two of the three initation sites accumulated similarly in cells infected with the ΔOriS virus or wild-type parent virus. The low-abundance transcript initiating from the third site was apparently unique to the ΔOriS virus but was not expected to alter the coding capacity of the mRNA. (iv) Infected cells accumulated RNA derived by antisense transcription of the genome domain containing the US11 gene. One transcript accumulated in larger amounts in cells infected with the ΔOriS virus than in cells infected with parent or repaired virus
Three-Dimensional Visualization of Tegument/Capsid Interactions in the Intact Human Cytomegalovirus

AbstractThe three-dimensional structure of the intact human cytomegalovirus (HCMV) was determined to 18-Å resolution by electron cryomicroscopy and computer reconstruction. Its capsid shell is composed of pentons, hexons, and triplexes arranged on a T = 16 icosahedral lattice and is identical to that of the B-capsid isolated from host cell nuclei. An icosahedrally ordered tegument layer formed by 960 copies of filamentous density is also visualized, which interacts with the pentons, hexons, and triplexes of the underlying capsid. The observed structural similarities and differences of HCMV with those of herpes simplex virus offer insights into the significance of the different tegument components for their infection processes while maintaining similar capsids