pinecone/core-2020-05-10-deduplication huggingface dataset:
A Functional Link between the Actin Cytoskeleton and Lipid Rafts during Budding of Filamentous Influenza Virions

AbstractMorphogenesis of influenza virus is a poorly understood process that produces two types of enveloped virion: ∼100-nm spheres and similar diameter filaments that reach 20 μm in length. Spherical particles assemble at plasma membrane lipid rafts in a process independent of microfilaments. The budding site of filamentous virions is hitherto uncharacterised but their formation involves the actin cytoskeleton. We confirm microfilament involvement in filamentous budding and show that after disruption of cortical actin by jasplakinolide, HA, NP, and M1 redistributed around β-actin clusters to form novel annular membrane structures. HA in filamentous virions and jasplakinolide-induced annuli was detergent insoluble at 4°C. Furthermore, in both cases HA partitioned into low buoyant density detergent-insoluble glycolipid domains, indicating that filamentous virions and annuli contain reorganised lipid rafts. We propose that the actin cytoskeleton is required to maintain the correct organisation of lipid rafts for incorporation into budding viral filaments
Clustered Charge-to-Alanine Mutagenesis of Human Respiratory Syncytial Virus L Polymerase Generates Temperature-Sensitive Viruses

AbstractClustered charge-to-alanine mutagenesis was performed on the large (L) polymerase protein of human respiratory syncytial virus to identify charged residues in the L protein that are important for viral RNA synthesis and to generate temperature-sensitive viruses. Clusters of three, four, and five charged residues throughout the entire L protein were substituted with alanines. A minigenome replicon assay was used to determine the functions of the mutant L proteins and to identify mutations that caused temperature sensitivity by comparing the level of reporter gene expression at 39 and 33°C. Charge-to-alanine mutations were introduced into an antigenomic cDNA derived from RSV A2 strain to recover infectious viruses. Of the 27 charge-to-alanine mutations, 17 recombinant viruses (63%) were obtained. Seven mutants (41%) exhibited small plaque morphologies and/or temperature-sensitive growth in tissue culture. To generate mutant viruses with more temperature-sensitive and attenuated phenotypes, several clusters of charge-to-alanine substitutions were combined. Five combination mutants were recovered that exhibited shut-off temperatures ranging from 36 to 39°C in tissue culture and restricted replication in the respiratory tracts of cotton rats
A Simian Immunodeficiency Virus Nef Peptide Is a Dominant Cytotoxic T Lymphocyte Epitope in Indian-Origin Rhesus Monkeys Expressing the Common MHC Class I Allele Mamu-A*02

AbstractThe precise measurement of epitope-specific cytotoxic T lymphocyte (CTL) responses in simian immunodeficiency virus (SIV)- and simian–human immunodeficiency virus (SHIV)-infected or vaccinated rhesus monkeys has been important in the evaluation of potential HIV vaccine strategies. This quantitation of CTL has been limited to date by the identification of only one dominant SIV/SHIV epitope in these monkeys. We have recently defined a Nef CTL epitope p199RY (YTSGPGIRY) that is recognized by CD8+ T lymphocytes from all SIV/SHIV-infected Mamu-A*02+ rhesus monkeys that have been evaluated. We now measure the frequency of p199RY-specific CD8+ T lymphocytes in the peripheral blood of these monkeys with quantitative precision, using MHC class I/peptide tetramer staining and peptide-stimulated interferon-γ Elispot assays. These epitope-specific CD8+ T lymphocytes are present at a very high frequency and represent a significant proportion of the entire SIV- or SHIV-specific CD8+ T lymphocyte population in SIV/SHIV-infected Mamu-A*02+ rhesus monkeys. Knowledge of this dominant CTL epitope should prove valuable in the evaluation of HIV vaccine strategies using this animal model
Interaction of Geminivirus Rep Protein with Replication Factor C and Its Potential Role during Geminivirus DNA Replication

AbstractGeminivirus DNA replication during the rolling-circle stage depends on the use of a DNA primer, a strategy poorly understood as compared with other eukaryotic viral systems that rely on RNA or protein as primers. Here we have used wheat dwarf virus (WDV) with the aim of elucidating the events leading to recruitment of cell factors at the replication origin. We have identified a novel interaction of WDV Rep, the replication initiation protein, with the large subunit of the wheat replication factor C complex (TmRFC-1). In other systems, the heteropentameric RFC clamp loader complex stimulates loading of DNA polymerase δ to the primer-template. Expression of TmRFC-1 is subjected to cell-cycle regulation, with a peak in early S-phase. We show that WDV Rep stimulates binding of recombinant TmRFC-1 to a model substrate containing a 3′-OH terminus and a WDV Rep-binding site. This was confirmed using cellular fractions enriched for wheat RFC complex, supporting the idea that, in addition to generating a 3′-OH terminus during initiation of DNA replication, WDV Rep could participate in the recruitment of RFC to the newly formed primer. We propose that this pathway may represent an initial event to facilitate the assembly of other replication factors, e.g., PCNA and/or DNA polymerase δ, a model that could also apply to other eukaryotic replicons, such as nanoviruses, circoviruses, and parvoviruses with a similar DNA replication strategy
SHIV-KB9 Infection of Rhesus Monkeys Does Not Always Cause Disease-Contribution of Host Immune Factors and Thymic Output

AbstractSimian-human immunodeficiency virus (SHIV) infection in the macaque is a model of HIV pathogenesis. KB9, a molecular clone of SHIV 89.6P, induced rapid and profound CD4+ T cell loss in rhesus monkeys, followed by partial recovery in some. We describe another clinical outcome after intravenous SHIV-KB9 inoculation in two of six infected rhesus macaques: lack of signs of immunodeficiency with sustained CD4+ T cell counts, despite virus load levels similar to those of the animals with CD4+ lymphocyte decline. To dissect the role of host factors in determining pathogenicity of this viral clone, humoral and cellular immune responses were studied. Differences in CD8+ T cell effector responses and activation profiles and in thymic output, but not in specific antibody production, were observed in animals with different disease outcomes during acute infection. Thymic involvement may thus be a critical factor in determining disease progression
Successful Protection by Amantadine Hydrochloride against Lethal Encephalitis Caused by a Highly Neurovirulent Recombinant Influenza A Virus in Mice

AbstractA mouse model system for a lethal encephalitis due to influenza has been established by stereotaxic microinjection with the recombinant R404BP strain of influenza A virus into the olfactory bulb of C57BL/6 mice. The virus infection spread selectively to neurons in nuclei of the broad areas of the brain parenchyma that have anatomical connections to the olfactory bulb, leading to apoptotic neurodegeneration. The inflammatory reaction at the extended stage of viral infection involved the vascular structures affected by induction of inducible nitric oxide synthase and protein nitration; those were related to the etiology of fatal brain edema. The intraperitoneal administration of amantadine inhibited the viral growth in the brain and saved mice from the lethal encephalitis. The severity of neuronal loss paralleled the time lag between the virus challenge and the start of amantadine treatment. Thus, early pharmacological intervention is essential to minimize neurological deficits due to influenza virus-induced neurodegeneration
Biochemical Characterization of Bacteriophage Lambda Genome Packaging in Vitro

AbstractBacteriophage λ has been extensively studied, and the abundance of genetic and biochemical information available makes this an ideal model system to study virus DNA packaging at the molecular level. Limited in vitro packaging efficiency has hampered progress toward this end, however. It has been suggested that limited packaging efficiency is related to poor activity of purified procapsids. We describe the construction of a vector that expresses λ procapsids with a yield that is 40-fold greater than existing systems. Consistent with previous studies, packaging of a mature λ genome is very inefficient in vitro, with only 4% of the input procapsids utilized. Concatemeric DNA is the preferred packaging substrate in vivo, and procapsids interact with a nucleoprotein complex known as complex I to initiate genome packaging. When complex I is used as a packaging substrate in vitro, capsid utilization is extremely efficient, and 40% of the input DNA is packaged. Finally, we provide evidence for a packaging-stimulated ATPase activity, and kinetically characterize this reaction quantifying the energetic cost of DNA packaging in bacteriophage λ
Transduction of Umbilical Cord Blood CD34+ NOD/SCID-Repopulating Cells by Simian Foamy Virus Type 1 (SFV-1) Vector

AbstractFoamy viruses are nonpathogenic retroviruses that offer unique opportunities for gene transfer into various cell types including hematopoietic stem cells. We used a simian foamy virus type 1 vector (SFV-1) containing a LacZ reporter gene with a titer of 1–5 × 106 viral particles/ml that was free of replication-competent retrovirus to transduce human umbilical cord blood CD34+ cells. Transduced CD34+ cord blood cells were transplanted into NOD/SCID mice and plated in serum-free methylcellulose culture to determine the transduction efficiency of human hematopoietic progenitor cells. A transduction efficiency of about 20% was obtained. At 6–10 weeks posttransplantation, human hematopoietic cell engraftment and marking were determined. Marrow from transplanted mice demonstrated human cell engraftment by the presence of human (CD45+) cells containing both CD19+ lymphoid and CD33+ myeloid cells. Serial sampling of NOD/SCID bone marrow revealed the presence of 6.7–14.0% CD45+ cells at 6 weeks posttransplant as compared to 3.6–27.2% CD45+ cells at 9–10 weeks posttransplant. Human progenitors examined from NOD/SCID bone marrow cells 9 weeks posttransplant revealed from 7.4 to 25.9% of the colonies exhibiting X-gal staining. Our study demonstrates the ability of a simian foamy virus vector to transduce the SCID-repopulating cell and offers a promising new gene delivery system for use in hematopoietic stem cell gene therapy
The Domain Order of Mammalian Capping Enzyme Can Be Inverted and Baculovirus Phosphatase Can Function in Cap Formation in Vivo

AbstractThe bifunctional mammalian mRNA capping enzyme (Mce1) consists of an N-terminal triphosphatase domain Mce1(1–210) fused to a C-terminal guanylyltransferase domain Mce1(211–597). The physical domain order H2N-triphosphatase-guanylyltransferase-COOH mimics the temporal order of the capping reactions. To determine if the physical domain order is functionally important in vivo, we engineered an “inverted” mammalian capping enzyme InvMce1 [H2N-Mce1(211–597)-(1–210)-COOH]. We found that InvMce1 complemented the growth of Saccharomyces cerevisiae cet1Δ and ceg1Δ strains in which the endogenous yeast triphosphatase and guanylyltransferase genes were deleted. By testing truncated versions of InvMce1, we determined that Mce1(1–178) comprises a minimal functional triphosphatase domain. Baculovirus phosphatase (BVP) is a monofunctional single-domain protein with RNA triphosphatase and RNA diphosphatase activities and an undefined role in viral RNA metabolism. Here we demonstrated that BVP can function as an RNA triphosphatase for cap formation in vivo when fused to the C-terminus of Mce1(211–597). By characterizing a series of InvMce1-BVP derivatives with amino acid substitutions in the phosphate-binding loop of BVP, we showed that the in vivo activity of the mutant chimeras in cap formation is contingent upon in vitro phosphohydrolase activity of the respective BVP proteins. BVP catalysis in vitro was not limited to 5′-phosphorylated RNA or nucleotide substrates, but also embraced tripolyphosphatase and pyrophosphatase activities. BVP-specific activities with nucleotide and inorganic substrates were as follows: ATP (14 min−1), ADP (31 min−1), PPPi (3.7 min−1), and PPi (1 min−1). BVP did not hydrolyze AMP. We surmise that BVP has adapted the cysteinyl phosphatase fold to the hydrolysis of phosphoanhydrides
Infection of BALB/c Mice with a Herpes Simplex Virus Type 1 Recombinant Virus Expressing IFN-γ Driven by the LAT Promoter

AbstractA recombinant herpes simplex virus type 1 expressing murine interferon-γ (IFN-γ) was constructed (HSV-IFN-γ) to study the effect of IFN-γ expression on HSV-1 infection of mice. HSV-IFN-γ was created by inserting the gene for murine IFN-γ under the control of the latency-associated transcript (LAT) promoter in a LAT-negative recombinant virus. ELISA analysis confirmed that the recombinant virus expressed high levels of IFN-γ in tissue culture. The recombinant HSV-IFN-γ had reduced virulence compared with the wild-type and LAT− parental strains as judged by death following ocular and ip infections in BALB/c mice. Replication of HSV-IFN-γ was wild type in tissue culture and mouse eyes. In addition, peak HSV-IFN-γ titers in mouse trigeminal ganglia (TG) and brain were similar for all viruses, although HSV-IFN-γ appeared in the TG and brains of ocularly infected mice earlier than either parental virus. Following stimulation with UV-inactivated virus, lymphocytes from HSV-IFN-γ-infected mice appeared to produce a steady level of interleukin-2 (IL-2) and IFN-γ throughout the first week of infection, while the IL-2 and IFN-γ levels in lymphocytes from wild-type and the LAT-negative parental virus, dLAT2903, varied over time. Also in contrast to lymphocytes from wild-type and dLAT2903-infected mice, lymphocytes from HSV-IFN-γ-infected mice produced no detectable IL-4. Following stimulation with recombinant IFN-γ (rIFN-γ), lymphocytes from HSV-IFN-γ-infected mice produced higher levels of IFN-γ, as compared to lymphocytes from control virus-infected mice. Finally, CTL and cell proliferation induced by HSV-IFN-γ were similar to those of both parental viruses. Thus, this report demonstrates that (i) HSV-IFN-γ had reduced neurovirulence, despite having enhanced replication in the TG of infected mice; (ii) HSV-IFN-γ did not enhance CTL activity above that seen in wild-type infected mice; and (iii) HSV-IFN-γ induced a TH1 pattern of cytokine response
Production of Hybrid Double- or Triple-Layered Virus-Like Particles of Group A and C Rotaviruses Using a Baculovirus Expression System

AbstractDual infections by group A and group C rotaviruses have been reported, but no reassortants between group A and group C rotaviruses have been described. The VP6 major inner capsid protein of group A and C rotaviruses shares common antigens detected by monoclonal antibodies and also shares 40–43% amino acid identity. Coinfection of Spodoptera frugiperda (Sf9) insect cells with different combinations of the recombinant baculoviruses encoding either group A {RF VP2 (A-VP2), IND VP6 (A-VP6), and VP7 (A-VP7[IND]), 2292B VP7 (A-VP7[2292B])} or C [Shintoku VP6 (C-VP6) and VP7 (C-VP7)] bovine rotavirus proteins produced hybrid group A/C triple-layered VP2/6/7 virus-like particles (TLPs) composed of A-VP2/C-VP6/C-VP7, A-VP2/C-VP6/A-VP7(IND), A-VP2/C-VP6/A-VP7(2292B), and A-VP2/A-VP6/C-VP7. To our knowledge, this is the first report that the inner capsid VP6 of group A or group C rotavirus can support attachment of the heterologous, antigenically distinct group A (G6, IND or G10, 2292B) or group C rotavirus outer capsid VP7 to produce hybrid TLPs in vitro
Enhanced Clearance of Herpes Simplex Virus Type 1 and Reduced Herpetic Eye Disease in STAT6 Knockout Mice Is Associated with Increased IL-2

AbstractSTAT6 (signal transducers and activators of transcription 6)-deficient (STAT6−/−) mice have defects in IL-4- and IL-13-mediated functions and thus have a reduced TH2-mediated immune response. Conversely, they have elevated levels of IL-2 and thus an increased TH1-mediated immune response. To assess the relative impact of reduced TH2- and elevated TH1-dependent immune responses on HSV-1 infection, vaccinated and mock-vaccinated STAT6−/− mice were challenged ocularly with HSV-1. Mock-vaccinated STAT6−/− mice were as susceptible to lethal HSV-1 infection as parental BALB/c mice. Mock-vaccinated STAT6−/− mice had reduced HSV-1 titers in their eyes compared to BALB/c mice. Furthermore, mock-vaccinated STAT6−/− mice had significantly less corneal scarring than their BALB/c counterparts. Vaccination induced significantly higher serum-neutralizing antibody titers in STAT6−/− mice compared to BALB/c mice, while completely protecting both types of mice against HSV-1-induced death and corneal scarring. Vaccinated STAT6−/− mice had reduced HSV-1 titers in their eyes compared to BALB/c mice. Lymphocytes from both vaccinated and mock-vaccinated STAT6−/− mice secreted higher amounts of IL-2 than lymphocytes from BALB/c mice, in the presence or absence of stimulation with UV-inactivated HSV-1. Finally, depletion of IL-2 increased ocular virus replication in STAT6−/− mice to levels similar to that measured in BALB/c mice. Our results suggest that in the absence of the STAT6 pathway, IL-2-mediated immune responses are up-regulated. This, in turn, leads to faster viral clearance and, consequently, lower levels of eye disease
Vpr- and Nef-Dependent Induction of RANTES/CCL5 in Microglial Cells

AbstractMicroglia are pivotal in the pathogenesis of AIDS dementia, as they serve as the major target of HIV infection in the CNS. In addition, activation of microglia correlates best with clinical dementia. Although the β-chemokine RANTES/CCL5 is important in modulating HIV infection as well as cellular activation, no information is available regarding how its expression is regulated in microglia by HIV-1. Here we report that RANTES/CCL5 expression is induced in microglia by HIV-1, but that this requires infection by HIV-1. This conclusion was supported by (1) the delayed kinetics coinciding with viral replication; (2) the lack of effect of X4 viruses; (3) inhibition by the reverse transcriptase inhibitor AZT, and (4) the lack of effect of cytokine antagonists or antibodies. Interestingly, RANTES/CCL5 production was dependent on the viral accessory protein Vpr, in addition to Nef, demonstrating a novel role for Vpr in chemokine induction in primary macrophage-type cells. Furthermore, the specific p38 MAP kinase inhibitor SB203580 augmented chemokine expression in microglia, indicating a negative role played by p38. These data suggest unique features of RANTES/CCL5 regulation by HIV-1 in human microglial cells
Characterization of the Human Cytomegalovirus UL75 (Glycoprotein H) Late Gene Promoter

AbstractGlycoprotein H (gH, UL75) of human cytomegalovirus (HCMV) is an essential envelope glycoprotein that functions in viral entry and the activation of gene expression. To understand the regulation of this important viral gene, the promoter of the UL75 late gene was characterized in HCMV-infected cells at the late stages of viral infection. Primer extension analysis revealed a single major start site located 26 bp downstream of a putative TATA element. Deletion analysis showed the presence of a dominant activation domain from +14 to +35 that masked regulatory sequences upstream of the TATA element. Mutational analysis demonstrated that a PEA3-like element in this downstream domain was important for promoter activation. In addition, gel shift analysis revealed direct protein binding to the PEA3-like element. Together, these studies reveal that the gH promoter is regulated in a complex manner with sequences both upstream and downstream of the cap site influencing promoter activation
Characterization of a Novel Human Endogenous Retrovirus, HERV-H/F, Expressed in Human Leukemia Cell Lines

AbstractWe have identified and characterized a human endogenous retrovirus (HERV) gag transcript in the human pre-B cell leukemia line Reh. The transcript was found to be a splice product of a structurally intact HERV element located on chromosome 6q13. Its primer binding site is complementary to phenylalanine (F) tRNA, common for the HERV-F family, but the overall genome sequence is closely related to the HERV-H family. The retroviral sequence was therefore designated HERV-H/F. The HERV element shows a distinct mRNA expression pattern among hematopoietic cancer cell lines with expression in some leukemia-derived cell lines of B-lymphoid and myeloid origin. No expression was observed in normal human tissues, indicating a cancer-specific expression pattern. The 5′ long terminal repeat (LTR) was tested for promoter activity in HERV-H/F expressing and nonexpressing cell lines. The cell specificity of the LTR-mediated reporter gene expression did not conclusively correlate with endogenous virus expression, indicating that the transcription regulation of this gene is not alone dependent on cell-specific activity of transcription factors
Rous Sarcoma Virus Negative Regulator of Splicing Selectively Suppresses src mRNA Splicing and Promotes Polyadenylation

AbstractRetroviruses require a balance of spliced and unspliced RNA for efficient replication. Here, we examined the effect of mutations in a splicing suppressor sequence called the negative regulator of splicing (NRS), located within the gag gene of Rous sarcoma virus. While the NRS mutant viruses showed only small changes in the levels of spliced env mRNAs, they had significant increases in src mRNA levels and transformed cells more efficiently than wild-type virus. None of these mutations prevented viral replication; however, some of the mutant viruses replicated more slowly than wild-type virus. In addition, increased transcriptional readthrough of the poly(A) site in the 3′ LTR was observed with the NRS mutant viruses, suggesting that the wild-type NRS sequence promotes polyadenylation
Expression of a Full-Length Hepatitis C Virus cDNA Up-Regulates the Expression of CC Chemokines MCP-1 and RANTES

AbstractWe had previously reported the cloning of the complete genome of an isolate of hepatitis C virus (HCV), HCV-S1, of genotype 1b. We have constructed a full-length complementary DNA (cDNA) clone of HCV-S1 using nine overlapping cDNA clones that encompassed its entire genome. HCV core, E1, E2, NS-3, -4B, -5A, and -5B proteins were detected in 293T cells by immunoblot analyses when expression of the full-length HCV-S1 was driven under a CMV promoter. Expression of full-length HCV-S1 led to induction of the CC chemokines RANTES and MCP-1 at both the mRNA and the protein levels in HeLa, Huh7, and HepG2 cells. Reporter gene assays showed that a minimal MCP-1 promoter construct containing 128 nucleotides upstream of its translational start site was sufficient for optimal HCV-mediated activation. HCV induced AP-1 binding activities to this region, as determined from electrophoretic mobility shift assays and supershifts with anti-AP-1 antibodies. Transfection of full-length HCV-S1 up-regulated both AP-1 binding activities as well as c-jun transcripts. A minimal promoter construct containing 181 nucleotides upstream of the RANTES translational start site was sufficient for maximal HCV-mediated induction. Gel mobility shift and supershift assays showed that HCV induced NF-κB and other unknown binding activities to the A/B-site within this region. In HeLa cells, HCV core and NS5A could separately augment promoter activities of both MCP-1 and RANTES. In Huh7 cells, only NS5A produced a similar effect, while rather surprisingly, HCV core induced a dramatic reduction in promoter activities of these two genes. This study provides the first direct evidence for the induction of CC chemokines in HCV infection and draws attention to their roles in affecting the progress and outcome of HCV-associated liver diseases