pinecone/core-2020-05-10-deduplication huggingface dataset:
Domain-Structure of Cytoplasmic Border Region Is Main Determinant for Palmitoylation of Influenza Virus Hemagglutinin (H7)

AbstractWe have shown previously that the length of cytoplasmic tails influences the selection of lipid substrates for palmitoylation of influenza viral hemagglutinin esterase fusion (HEF) and hemagglutinin (HA) glycoproteins [Veitet al.(1996)Biochem. J.318, 163–172; Revereyet al.(1996)J. Biol. Chem.271, 23607–23610]. Using a series of new chimeric mutant proteins derived from acylated influenza virus HA (subtype H7) and from nonacylated Sendai virus fusion protein (F, strain Z), we report here that palmitoylation levels depend on the type of transmembrane or cytoplasmic domain, or both, present in the expression products and that cysteine residues placed close to the cytoplasmic membrane border are not sufficient for acylation. By inserting stretches of the HA transmembrane domain into a nonacylated mutant of Sendai F (FCys), we induce palmitoylation after expression in CV.1 cells, and the level of fatty acid transfer increases with the length of the HA-derived insert. A five-amino-acid shift of the HA transmembrane domain severely augments fatty acid transfer. Our data suggest that the influenza virus HA contains complex conformational signals for palmitoylation that are mainly located within the transmembrane domain but also involve the C-tail region, whereas the extracellular (luminal) domain has only marginal influence on palmitoylation
Shope Fibroma Virus RING Finger Protein N1R Binds DNA and Inhibits Apoptosis

AbstractShope fibroma virus (SFV) N1R gene encodes a RING finger protein that localizes to virus factories within the cytoplasm of infected cells. Altered proteins, with deletions and site-specific mutations, were transiently expressed in vaccinia virus-infected cells to discern regions of the protein that are required for localization. We have determined that at least part of the RING finger region is necessary for localization but that the RING motif alone is not sufficient. A chimeric protein, however, in which the RING finger region of the herpes simplex virus-1 ICP0 protein replaces the SFV N1R RING motif does localize to virus factories. A region of five highly conserved amino acids at the amino terminus of SFV N1R is also critical for localization. We report that the SFV N1R protein binds double- and single-stranded DNA, suggesting a mechanism for localization, and that overexpression of this protein in vaccinia virus-infected cells reduces apoptosis-associated fragmentation of nuclear DNA
Analysis of Human Herpesvirus 6 U3 Gene, which Is a Positional Homolog of Human Cytomegalovirus UL 24 Gene

AbstractThe US22 gene family was first discovered in human cytomegalovirus (HCMV) and contains several conserved amino acid motifs. Human herpesvirus 6 (HHV-6) also encodes several genes belonging to the US22 family, including the U3 gene (a positional homolog of HCMV UL24). Because the gene products of the US22 gene family function as gene regulators in general, we analyzed the HHV-6A U3 gene. Six transcripts with different molecular weights of 7.5–1.8 kb were detected by Northern blot analysis using a U3-specific probe. Sequence analyses of the respective cDNA clones and primer extension experiments revealed that the U3 gene encoded the 2.0- and 3.5-kb transcripts and had no splicing within the U3 gene region. By immunofluorescence testing using antibodies raised to a fusion protein of MBP (maltose-binding protein) and U3, the U3 viral antigen was detected as early as 24 h p.i. in HHV-6A-infected U373 cells. The antigens were found in cytoplasmic granules, preferentially in the endoplasmic reticulum. Moreover, cotransfection assay using a luciferase gene expression system revealed that the U3 gene product was capable of activating the human immunodeficiency virus long terminal repeat promoter in CV1 cells
Use of GPR1, GPR15, and STRL33 as Coreceptors by Diverse Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus Envelope Proteins

AbstractHuman and simian immunodeficiency viruses (HIV and SIV, respectively) use chemokine receptors as coreceptors along with CD4 to mediate viral entry. Several orphan receptors, including GPR1, GPR15, and STRL33, can also serve as coreceptors for a more limited number of HIV and SIV isolates. We investigated whether these orphan receptors could function as efficient coreceptors for a diverse group of HIV and SIV envelopes (Envs) in comparison with the principal coreceptors CCR5 and CXCR4. We found that a limited number of HIV-1 isolates could mediate inefficient cell–cell fusion with the orphan receptors relative to CCR5 and CXCR4; however, none of the orphan receptors tested could support pseudotype virus infection despite robust infection via CCR5 or CXCR4. All except one of the SIV Envs tested mediated some degree of cell–cell fusion and pseudotype infection, with target cells expressing at least one of these orphan receptors, although CCR5 proved to be the most efficient coreceptor for infection. Only one SIV Env protein, BK28, could mediate infection using GPR1 as a coreceptor, albeit much less efficiently than with CCR5. In addition, use of these coreceptors did not correlate with the published tropism of the SIV clones and was strictly CD4 dependent for both SIV and HIV. We also examined the expression of these molecules in cell lines and primary cells widely used for virus propagation and as targets for infection. All cells examined expressed STRL33, a more limited number expressed GPR15, and GPR1 was much more restricted in its expression pattern. Taken together, our results indicate that GPR15 and STRL33 are rarely used by HIV-1 but are more frequently used by SIV strains, although not in a manner that correlates with SIV tropism
Dominant Host Range Selection of Vaccinia Recombinants by Rescue of an Essential Gene

AbstractWe report the rescue of a defective vaccinia virus, forming the basis for a stringent selection protocol to generate replicating recombinant virus without the need for marker cassettes and selection agents. Plaques of recombinant virus could be isolated solely by their ability to grow in wild-type cells normally supporting the growth of vaccinia virus. All growth-competent clones analyzed contained the gene of interest in the intended genomic locus and displayed foreign gene expression to the same levels as was seen with classical recombinants obtained by insertion into the vaccinia virus thymidine kinase locus. The system is based on a defective vaccinia virus, expressing exclusively early genes, termed eVAC-1, and an insertion plasmid vector providing the essential function, the uracil DNA glycosylase gene. In addition, the defective virus is free of selection and color marker genes, thus also representing a basic vector for the generation of defective recombinants
Intracellular Virus DNA Distribution and the Acquisition of the Nucleoprotein Core during African Swine Fever Virus Particle Assembly: Ultrastructuralin SituHybridisation and DNase-Gold Labelling

AbstractAfrican swine fever virus (ASFV) is a large complex icosahedral double-stranded DNA virus that replicates in the cytoplasm of susceptible cells. Assembly of new virus particles occurs within the perinuclear viroplasm bodies known as virus factories. Two types of virus particle are routinely observed: “fulls,” which are particles with an electron-dense DNA-containing nucleoid, and “empties,” which consist of the virus protein and membrane icosahedral shell but are without the incorporation of the virus genome. The objective of this study was to understand ASFV morphogenesis by determining the distribution of intracellular viral DNA in the virus factory and during virus particle assembly. The ultrastructural localisation of DNA within ASFV-infected cells was achieved using two complementary methods: with an ASFV-specific DNA probe to the major capsid protein (p73) gene (B646L) hybridisedin situor through detection of all forms of DNA (viral and cellular) with gold-labelled DNase. Conditions forin situhybridisation at the electron microscopic level were optimised for infected cells in two Lowicryl resins (K4M and HM20) and using two nonradioactive probe labels (digoxygenin and biotin). The morphological data indicate that the viral DNA, perhaps from specialised storage sites within the factory, begins to condense into a pronucleoid and is then inserted, at a single vertex, into an “empty” particle. Further maturation of the viral particle, including closure of the narrow opening in the icosahedron, gives rise to “intermediate” particles, where the nucleoprotein core undergoes additional consolidation to produce the characteristic mature or “full” virions. The site of particle closure may represent a “weak point” at one vertex, but the mechanisms and structures involved in the packaging and release of the virus genome via such a port are yet to be determined
Pathogenesis of Murine Gammaherpesvirus-68 Infection in Interleukin-6-Deficient Mice

AbstractMurine gammaherpesvirus-68 (MHV-68) induces high levels of interleukin (IL)-6 production in both naive and primed lymphocyte populations. Mice that are homozygous (−/−) for deletion of the IL-6 gene were used to investigate the role of this cytokine in MHV-68 infection. The results showed that IL-6 is not essential for clearance of infectious MHV-68 from the lung or for the establishment, or control, of viral latency. Both IL-6 +/+ and −/− mice eliminated replicating virus from the respiratory tract within 15 days of infection, and their lungs remained clear of infectious virus for ≥150 days. Interestingly, the IL-6 −/− mice had both increased numbers of natural killer (NK)1.1+ cells and higher levels of NK cell activity than the +/+ controls at 10–15 days after infection. However, there was no difference in the cytotoxic T cell activity between the two groups of mice. Levels of latent virus were comparable in IL-6 +/+ and −/− mice over the time course studied. Furthermore, analysis of the numbers, types, and activation status of the various leukocyte subsets (other than NK cells) in the bronchoalveolar lavage population, lymph nodes, and spleens of +/+ and −/− mice revealed no striking differences. Apart from the expected lack of IL-6, cytokine profiles were not dramatically altered in IL-6 −/− mice. Thus, there is no evidence for an obligatory role for IL-6 in T cell activation during infection with MHV-68
Assembly of Hepatitis Delta Virus Particles: Package of Multimeric Hepatitis Delta Virus Genomic RNA and Role of Phosphorylation

AbstractWe previously demonstrated that both casein kinase II (CKII) and protein kinase C (PKC) positively modulate the hepatitis delta virus (HDV) RNA replication but not the assembly of the empty hepatitis delta antigen (HDAg) particle. In this study, we investigated whether phosphorylation of HDAg by these two kinases plays a role in assembly of the HDV virion. As demonstrated byin vivolabeling and kinase inhibitor experiments, the phosphorylation level of large HDAg but not small HDAg in HDAg-expressing HuH-7 cells was diminished by CKII inhibitor (DRB), whereas no effect was observed for the phosphorylation level of two HDAgs when treated with protein kinase A (PKA) inhibitor (HA1004) or PKC inhibitor (H7). Cotransfection experiment also demonstrated that packaging of HDV genomic RNA was not affected by the kinase inhibitor DRB or H7 and mutation at the putative CKII phosphorylation sites (serine-2, serine-123, or both), and the putative PKC site (serine-210) of HDAg did not elicit any significant effect on the HDV virion assembly. Therefore, based on the previous work and the present study, it seems that the status and biological significance of phosphorylation of HDAg vary depending on the HDV life cycle. Although in the HDV RNA replication cycle, phosphorylation of small HDAg by CKII or PKC plays important role in HDV replication, phosphorylation of the same HDAg by these two kinases does not occur during the HDV RNA virion assembly, and phosphorylation of the large HDAg by CKII does not confer any regulatory role in the assembly of HDV virion and empty viral particles. Our study also showed that the large HDAg without the small HDAg could efficiently assemble both monomeric and dimeric HDV genomic RNAs into secreted HBV-enveloped virus-like particles. Increasing the transfected small HDAg-expressing plasmid led to an enhancement of the packaging efficiency for the monomeric HDV genomic RNA with little effect on the packaging of dimeric HDV RNA. Similarly, HDAgs could package the trimeric HDV genomic RNA, albeit less efficiently. CsCl density gradient centrifugation confirmed that HDAgs and the monomeric and multimeric (dimer and trimer) HDV genomic RNAs formed an HBV-enveloped virus-like particle at a density of 1.23–1.25 g/ml. Thus, the assembly of the HDV virion seems to not impose much restriction on the size of HDV RNA for packaging
Biochemical and Kinetic Analyses of NS5B RNA-Dependent RNA Polymerase of the Hepatitis C Virus

AbstractThe biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagged NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of the purified protein was inhibited by KCl or high concentrations of NaCl and was absolutely dependent on Mg2+, which could be replaced by Mn2+. NS5B was found to be processive and able to copy long heteropolymeric templates with an elongation rate of 150–200 nucleotides/min at 22°C. Kinetic constants were determined for all four nucleoside triphosphates and different templates. In case of a heteropolymeric RNA template corresponding to the last 319 nucleotides of the hepatitis C virus genome,Kmvalues for UTP, GTP, ATP, and CTP were ∼1.0, ∼0.5, ∼10, and ∼0.3 μM, respectively. The profile of several inhibitors of RdRp activity and substrate analogs indicated that the enzyme has a strong preference for ribonucleoside 5′-triphosphates and that it closely resembles 3Dpolof picornaviruses
Effect of Rev on the Intranuclear Localization of HIV-1 Unspliced RNA

AbstractHuman immunodeficiency virus type 1 (HIV-1) Rev is a 19-kDa regulatory protein which binds to unspliced and partially spliced HIV-1 RNAs. Export, splicing, stability, and translation of HIV-1 RNAs are influenced by Rev. To further understand the effect of Rev on HIV-1 RNA splicing, the intranuclear localization of unspliced HIV-1 RNA and a cellular splicing factor was examined in the presence and absence of Rev. Splicing component-35 (SC-35) is an essential SR protein splicing factor which localizes into 20–40 nuclear granules (Fu, X. D., and Maniatis, T.Nature343 (6257), 437–441, 1990). Laser scanning confocal microscopy was utilized to examine the colocalization of unspliced HIV-1 RNA and SC-35-containing granules. In the presence of Rev, many of the SC-35-containing granules were colocalized on their edges or completely colocalized with HIV-1 unspliced RNA speckles. In the absence of Rev, however, little colocalization of the unspliced HIV-1 RNA speckles and the SC-35-containing granules was observed. Quantitative RT–PCR was utilized to examine the effect of Rev on the level of fully spliced HIV-1 RNA. In the presence of Rev, a decrease in the level of fully spliced HIV-1 RNA was observed. Thus both the intranuclear localization and posttranscriptional processing of HIV-1 unspliced RNA are affected by Rev
Antigenic Variation of Varicella Zoster Virus Fc Receptor gE: Loss of a Major B Cell Epitope in the Ectodomain

AbstractVaricella zoster virus (VZV) is considered to possess a genetically stable genome; only one serotype is recognized around the world. The 125-kbp genome contains ∼70 open reading frames. One that has received particular attention is open reading frame 68, which codes for glycoprotein gE, the predominant 623-residue viral envelope product that harbors both B and T cell epitopes. This report describes the initial characterization of a community-acquired VZV isolate that was a distinguishable second serotype (i.e., it had lost a major B cell epitope defined on the gE ectodomain by a murine monoclonal antibody called mAb 3B3). The mAb 3B3 epitope was found not only on the prototype sequenced Dumas strain from Holland and all previously tested North American isolates but also on the varicella vaccine Oka strain originally attenuated in Japan. Sequencing of the mutated gE ectodomain demonstrated that codon 150 exhibited a single base change that led to an amino acid change (aspartic acid to asparagine). Observation of the monolayers infected with the mutant VZV strain also led to the surprising discovery that the topography of egress was altered. Wild-type VZV emerges along distinctive viral highways, whereas the mutant strain virions were nearly uniformly distributed over the cell surface in a pattern more closely resembling egress of herpes simplex virus 1. The mutant VZV strain was designated VZV-MSP because it was isolated in Minnesota
Cytodifferentiating Agents Affect the Replication of Herpes Simplex Virus Type 1 in the Absence of Functional VP16

AbstractThe herpes simplex virus type 1 (HSV-1) mutantin1814 encodes an altered form of the virion protein VP16 that is unable to transactivate immediate-early (IE) transcription. As a consequence of the mutation,in1814 initiates productive replication inefficiently after infection of tissue culture cells. Previous studies showed that this defect could be overcome by the inclusion in the culture medium of hexamethylene bisacetamide (HMBA), a compound that promotes the differentiation of murine erythroleukemia cells (MELCs). The effects of additional agents known to induce differentiation of MELCs were investigated.N′-Methylnicotinamide, at concentrations optimal for the induction of MELCs, complemented the replication ofin1814 and stimulated IE gene expression. Suberoyl bishydroxamic acid and suberoylanilide hydroxamic acid, which induce differentiation of MELCs at micromolar concentrations, did not complementin1814 but specifically blocked the action of HMBA. The histone deacetylase inhibitor trichostatin A, which also induces differentiation of MELCs, antagonized the effect of HMBA in a manner similar to that of suberoyl bishydroxamic acid and suberoylanilide hydroxamic acid. The results demonstrate that the requirement for VP16 activity is dependent on the metabolic state of the host cell and that the pathways leading to complementation ofin1814 and differentiation of MELCs are overlapping but not identical
Human Herpesvirus-8 ORF K8.1 Gene Encodes Immunogenic Glycoproteins Generated by Spliced Transcripts

AbstractA cDNA library from phorbol ester-induced human herpesvirus-8 (HHV-8) carrying BCBL-1 cells was screened with an HIV+KS+ serum, and several cDNA clones encoding HHV-8 proteins were identified. Sequence analysis of two full-length cDNA clones show open reading frames (ORFs) encoded by spliced messages originating from the HHV-8 K8.1 gene. One cDNA encodes an ORF of 228 amino acids, designated K8.1.A, with a cleavable signal sequence, a transmembrane domain, and fourN-glycosylation sites. The splicing event generated the transmembrane domain in the ORF not seen in the genomic K8.1 ORF. Another cDNA encodes an ORF of 167 amino acids, designated K8.1.B, that shares similar amino and carboxyl termini with ORF K8.1.A but with an in-frame deletion. The primary translation products of ORF K8.1A (34 kDa) and K8.1B (20 kDa) in thein vitro–transcription–translation experiments shifted into glycosylated species of 43 and 32 kDa, respectively, in the presence of microsomal membranes. This suggested that the ORF K8.1A and K8.1B encode for glycoproteins. Riboprobes from the K8.1A cDNA insert hybridized with an HHV-8-specific 0.9-kb abundant transcript from BCBL-1 cells. Synthesis of this RNA was eliminated in the presence of a DNA synthesis inhibitor, suggesting that this RNA was a late gene transcript. Because ORFs K8.1A and K8.1B are unique for HHV-8, human sera were tested in Western blot reactions for antibodies against glutathione-S-transferase–ORF K8.1A fusion protein. All sera that were positive for HHV-8 antibodies in immunofluorescence assays with phorbol ester-induced BCBL-1 cells were also positive for anti-ORF K8.1A antibodies. This suggests that measurement of anti-ORF K8.1A antibodies would provide an HHV-8-specific serological assay. Further work is needed to define the biological role of the HHV-8 ORF K8.1A and K8.1B glycoproteins
The V Protein of the Paramyxovirus SV5 Interacts with Damage-Specific DNA Binding Protein

AbstractThe simian parainfluenza virus 5 (SV5) V/P gene encodes two proteins: V and the phosphoprotein P. The V and P proteins are amino coterminal for 164 residues, but they have unique carboxyl termini. The unique carboxyl terminus of V contains seven cysteine residues, resembles a zinc finger, and binds two atoms of zinc. In a glutathione-S-transferase (GST)-fusion protein selection of cell lysate assay, the GST-V protein was found to interact with the 127-kDa subunit (DDB1) of the damage-specific DNA binding protein (DDB) [also known as UV-damaged DNA binding protein (UV-DDB), xeroderma pigmentosum group E binding factor (XPE-BF), and the hepatitis B virus X-associated protein 1 (XAP-1)]. A reciprocal GST-DDB1 fusion protein selection assay of SV5-infected cell lysates showed that DDB1 and V interact, and it was found that V and DDB1 could be coimmunoprecipitated from SV5-infected cells or from cells expressing V and DDB1 using the vaccinia virus T7 expression system. The interaction of V and DDB1 involves the carboxyl-terminal domain of V in that either deletion of the V carboxyl-terminal domain or substitution of the cysteine residues (C189, C193, C205, C207, C210, C214, and C217) in the zinc-binding domain with alanine was able to disrupt binding to DDB1. The V proteins of the mumps virus, human parainfluenza virus 2 (hPIV2), and measles virus have also been found to interact with DDB1 in GST-fusion protein selection assays usingin vitrotranscribed and translated DDB1
DnaA-Mediated Regulation of Phage λ-Derived Replicons in the Absence ofpRand Cro Function

AbstractBacteriophage λ-derived replicons can replicate inEscherichia colicells as plasmids. In the control of replication of these plasmids, an important role was ascribed to the λ Cro repressor autoregulatory loop. However, theoR/pR-cro-tR-cII′ region could be replaced by theptetApromoter under the control of the TetR repressor, producing plasmid pTCλ. Here, we demonstrate that stable maintenance of pTCλ depends on the host DnaA function because deletion of one of DnaA-binding sequences present in pTCλ resulted in a decrease in the plasmid (pTCλ) copy number and poor maintenance of pTCλ inE. coli. Moreover, in contrast to the replication of the wild-type λ plasmid, previously found to be positively regulated by DnaA (acting on a relaxed DnaA box situated immediately downstream of thepRpromoter), the replication of pTC plasmids (devoid ofpR) was found to be negatively regulated by DnaA. Contrary to wild-type λ plasmids, in cells harboring λcro[temperature-sensitive (ts)] or pTCλ (but not pTCλ) plasmid, the λ replication complex was heat shock resistant; this complex, however, disassembled after inactivation of DnaA function. This disassembly was blocked by DNA gyrase inhibitors. According to our model outlined previously, we propose that the heat shock resistance of the replication complex of λcro−plasmids depends on the interaction of the DNA-bound DnaA protein with the DNA-bound λ replication complex. The replication complex-DnaA-λ DNA structure may be directly related to the role of DnaA as the Cro-replacing negative regulator of λcro−plasmid replication