pinecone/core-2020-05-10-deduplication huggingface dataset:
Proteolytic Activity of Purified Avian Sarcoma and Leukemia Virus NC-PR Protein Expressed inEscherichia coli

AbstractProcessing of the internal structural and enzymatic proteins of retroviruses occurs during or shortly after budding and is accomplished by the viral protease (PR), which belongs to the large family of aspartic proteases. It is not known how the activity of PR is regulated so that proteolysis occurs at this time. Cellular aspartic proteases are synthesized as zymogens with short N-terminal extensions that are proteolytically removed to generate the free active enzyme. In the avian sarcoma and leukosis viruses (ASLV), PR is expressed as the carboxy-terminal domain of the Gag polyprotein, which thus has a structure analogous to such a zymogen. We have investigated the enzymatic properties of ASLV PR when it is part of a longer protein, NC-PR, serving as a model for Gag. This protein represents about one-third of Gag and consists of the nucleocapsid (NC) domain fused to the N-terminus of PR. NC-PR and derivatives of NC-PR were expressed in bacterial cells and purified. In short-term assays, these fusion proteins lacked measurable protease activity toward an exogenous substrate prepared byin vitrotranslation. In contrast to PR, which is a homodimer, NC-PR migrated as a monomer both by glycerol gradient sedimentation and by gel filtration chromatography. Thus the NC domain appears to inhibit enzymatic activity by altering the dimerization potential of the PR domains. However, upon long incubations NC-PR was found to cleave itself to generate free and fully active PR, implying that dimerization was not prevented entirely. On the basis of these results, we hypothesize that the Gag proteinin vivois also incompletely active as a protease, because upstream portions of Gag interfere with proper interaction of the PR domains. The eventual dimerization, perhaps triggered by other events, then could lead to a cascade whereby PR is proteolytically freed from Gag and thereby gains enzymatic activity
Sequence Analysis of a Highly Divergent HIV-1-Related Lentivirus Isolated from a Wild Captured Chimpanzee

AbstractTwo strains of simian immunodeficiency viruses (SIV) isolated from chimpanzees (SIVCPZ-GABand SIVCPZ-GAB2) originating from Gabon have previously been genetically characterized and shown to belong phylogenetically to the same lineage as the human immunodeficiency virus type 1 (HIV-1). We describe the sequence analysis of a third HIV-1-related virus, SIVCPZ-ANT, isolated from a wild captured chimpanzee originating from Zaire. This virus displayed the same genetic organization as HIV-1 and was found to fall on the same lineage as HIV-1 and SIVCPZ-GAB. Protein sequence identity with SIVCPZ-GABranged from 72% (Pol) to 48% (Env) for the structural proteins, while a particularly divergent Vpu was found (only 25% identity to SIVCPZ-GAB). The V3 regions of the SIVCPZisolates were exceptionally conserved in contrast to the high divergence of V3 among HIV-1 isolates. However, SIVCPZ-ANTdid not show a greater degree of sequence similarity with SIVCPZ-GABthan with HIV-1 isolates and represents a quite divergent outgroup of the HIV-1 lineage. Our data suggest multiple introductions of HIV-1 in the human population and shed new light on the origin of the HIV-1 pandemic
SFV Topoisomerase: Sequence Specificity in a Genetically Mapped Interval

AbstractPoxviral DNA topoisomerases are sequence-specific enzymes whose activities are thought to influence such diverse processes as transcription, DNA replication, and genetic recombination. To obtain further insights into the relatedness of these enzymes, and their influence on virus-mediated recombination, we have determined the target-specificity and other catalytic properties of the Shope fibroma virus (SFV) topoisomerase. SFV topoisomerase was expressed inEscherichia coliand purified as a glutathioneS-transferase (GST) or (his)6-tagged fusion protein. The recombinant Leporipoxvirus (SFV) enzyme displayed catalytic properties very similar to vaccinia topoisomerase. In particular SFV topoisomerase recognizes the same pentanucleotide motif [5′-(C/T)CCTT-3′] and promotes the same DNA relaxation, strand transfer, and strand cleavage reactions catalyzed by the Orthopoxviral (vaccinia) enzyme. The SFV enzyme can also efficiently cleave DNA 3′ of the variant site 5′-CCCTG-3′ in certain sequence contexts. These studies identified several sites where SFV topoisomerases interact with a recombinational substrate and permitted a comparison of recombination frequencies across intervals which did, or did not, span these sites. We failed to detect any effect of topoisomerase-recognition sites on recombination frequencies, except for a small (<2-fold) stimulation seen when the substrates encoded a nearby poxviral promoter. This and other work shows that poxviral topoisomerases from several genera share common target specificities, but other enzymatic systems probably catalyze the high-frequency recombination seen in poxvirus-infected cells
Identification of a Variable Region at the Carboxy Terminus of SV40 Large T-Antigen

AbstractThe entire early regions of three human isolates of simian virus 40 (SV40), as well as two laboratory strains recovered from monkeys, were sequenced. The early coding region of each isolate contains a number of nucleotide differences when compared to the reference strain SV40-776. These differences result in some changes in the predicted amino acid sequence of the unique region of small t-antigen and in the carboxy (C) terminus of large T-antigen. The amino acid sequence of the remainder of large T-antigen was absolutely conserved among all isolates. Thus, SV40 large T-antigen contains a variable domain at the C-terminal end of the molecule
Isolation and Characterization of Marek's Disease Virus (MDV) cDNAs from a MDV-Transformed Lymphoblastoid Cell Line: Identification of an Open Reading Frame Antisense to the MDVEco-Q Protein (Meq)

AbstractTwo Marek's disease virus (MDV) cDNAs of 852 and 1168 bp, which map to the right end of theBamHI-I2fragment of the MDV genome, were isolated from a cDNA library derived from the MDV transformed lymphoblastoid cell line MKT-1. These cDNAs hybridized to relatively abundant leftward mRNA transcripts in MKT-1 cells and cells lytically infected with MDV. The transcriptional initiation site for these transcripts was located in the adjacentBamHI-Q2fragment, as determined by RNase protection and primer extension assays. A computer search for the presence of leftward open reading frames (ORFs) revealed two ORFs encoding 135- and 195-amino-acid polypeptides. A polyclonal antibody raised against a protein sequence in the N-terminus of the latter ORF detected a 23-kDa protein in the nuclear fraction of MDV-transformed lymphoblastoid cells. Furthermore, this ORF was antisense to part of the MDVEco-Q protein (Meq) sequence
Infection and Cellular Activation by Human T-Cell Leukemia Viruses, Types I and II

AbstractResting peripheral blood mononuclear cells (PBMC) or purified T-cells can be induced to proliferate when coculturedin vitrowith fixed HTLV-infected T-cells. This process of HTLV-dependent cellular activation and induction of proliferation has been considered distinctive because of an apparent independence from conventional T-cell costimulatory signals. We have examined several HTLV-infected cell lines and found that proliferation was readily induced in resting PBMC by T-cells that were productively-infected with HTLV. However, equivalent HTLV-productive infection in a B-cell line failed to induce proliferation in PBMC, suggesting that HTLV-dependent induction of proliferation in PBMC was, at least in part, dependent upon a T-cell-specific signal. Furthermore, the induction of proliferation in PBMC populations was found to overlap with, and actually require, transfer and establishment of HTLV infection within the T-cell compartment of the PBMC population. These findings suggest that virus-induced activation of target cells may be directly associated with transfer and spread of HTLV infection
Comparison of the EBNA1 Proteins of Epstein–Barr Virus and Herpesvirus Papio in Sequence and Function

AbstractThe EBNA1 protein of Epstein–Barr virus (EBV) supports replication and maintenance of the circularized viral chromosome in cells that are latently infected. We have isolated, sequenced, and functionally characterized the EBNA1 gene of herpesvirus papio (HVP), an EBV-like virus that infects baboons. The amino acid sequences of EBNA1 of HVP and EBV are 56% identical, if the difference in the length of the glycine and alanine containing repetitive region, which is much shorter for HVP EBNA1, is omitted for the calculation. The key structural features of the DNA-binding/dimerization domain (the carboxyl-terminal domain) appear to have been conserved, as have amino acids in the two regions thought to be most critical for DNA binding. Most of the salient features of the amino-terminal two-thirds of EBNA1 (the amino-terminal domain), including a dearth of sequences predictive of alpha-helical or beta-sheet structures, are shared by the two sequences, although numerous gaps in this region were needed for alignment of the sequences. The amino-terminal fifty amino acids of EBNA1 of both EBV and HVP weakly resemble the amino terminus of rat ribosomal protein S2. Plasmids carryingoriPof either virus replicated stably in mammalian cells and supported efficient outgrowth of colonies under selection when supported by EBNA1 from either virus, although with eachoriPthere was a noticeable preference for EBNA1 to be from the same virus. HVP EBNA1 was less effective than EBV EBNA1 at activating the enhancer function of EBVoriPand under certain conditions was less effective than EBV EBNA1 at supporting maintenance of plasmids carrying EBVoriP.Results obtained with hybrid EBNA1 molecules indicated that differences in the amino-terminal and carboxyl-terminal domains, respectively, are primarily responsible for the differences in transcriptional activation and plasmid maintenance, respectively. The results showed that changes within EBNA1 can differentially alter its transcriptional and replicational activities
Identification and Deletion of Sequences Required for Feline Leukemia Virus RNA Packaging and Construction of a High-Titer Feline Leukemia Virus Packaging Cell Line

AbstractSequences required for specific encapsidation of feline leukemia virus (FeLV) genomic RNA have not yet been defined. Deletion of 107 nucleotides between the splice donor (SD) and thegagcoding region of a prototypic subgroup A FeLV, 61E, resulted in an approximately 200-fold reduction of packaged viral RNA. Virus particle production was not disrupted by the deletion, although viral infectivity was dramatically reduced. These data indicate that the 107-nucleotide sequence comprises a portion of the FeLV packaging signal. FeLV particles expressed from the deleted genome were able to efficiently package murine leukemia virus vectors, resulting in high-titer G418Rvirus production. This system can be easily adapted to produce FeLV particles that contain envelope proteins from other feline leukemia virus subgroups and will be broadly useful for studies of FeLV envelope/receptor interactions
Restrictive Type of Replication of Ovine/Caprine Lentiviruses in Ovine Fibroblast Cell Cultures

AbstractCaprine arthritis–encephalitis virus (CAEV) is a natural lentivirus pathogen of goats. CAEV, like all members of the ovine/caprine lentivirus family, has anin vivotropism for cells of the monocyte/macrophage cell lineage and activation of viral gene expression is observed only following differentiation of monocytes to macrophages. In addition to cells of the monocyte/macrophage lineage, CAEV and the closely related maedi visna virus of sheep (MVV) can also replicate productively in fibro-epithelial cells derived from synovial membrane of goats (GSM). However, these viruses varied greatly in their ability to replicate in fibroblasts. We studied the biological and biochemical properties of CAEV and maedi-visna virus (MVV) of sheep following inoculation into the three ovine/caprine cell types. Our data showed no substantial differences in virus titers, viral protein biosynthesis, or processing of the viral proteins between CAEV and MVV following inoculation into primary macrophages and GSM cells. However, unlike MVV, CAEV failed to replicate productively in ovine fibroblasts (sheep choroid plexus cells). This correlated with a specific but abnormal proteolytic cleavage of the envelope glycoprotein of the virus. This abnormal proteolytic cleavage represents a novel type of host cell restriction of lentivirus replication
Structural Requirements for Initiation of Translation by Internal Ribosome Entry within Genome-Length Hepatitis C Virus RNA

AbstractCap-independent translation of hepatitis C virus (HCV) RNA is mediated by an internal ribosomal entry segment (IRES) located within the 5′ nontranslated RNA (5′NTR), but previous studies provide conflicting views of the viral sequences which are required for translation initiation. These discrepancies could have resulted from the inclusion of less than full-length 5′NTR in constructs studied for translation or destabilization of RNA secondary structure due to fusion of the 5′NTR to heterologous reporter sequences. In an effort to resolve this confusion, we constructed a series of mutations within the 5′NTR of a nearly full-length 9.5-kb HCV cDNA clone and examined the impact of these mutations on HCV translationin vitroin rabbit reticulocyte lysates and in transfected Huh-T7 cells. The inclusion of the entire open reading frame in HCV transcripts did not lead to an increase in IRES-directed translation of the capsid and E1 proteins, suggesting that the nonstructural proteins of HCV do not include a translational transactivator. However, in reticulocyte lysates programmed with full-length transcripts, there were multiple aberrent translation initiation sites resembling those identified in some picornaviruses. The deletion of nucleotides (nt) 28–69 of the 5′NTR (stem-loop IIa) sharply reduced capsid translation bothin vitroandin vivo.A small deletion mutation involving nt 328–334, immediately upstream of the initiator AUG at nt 342, also resulted in a nearly complete inhibition of translation, as did the deletion of multiple intervening structural elements. An in-frame 12-nt insertion placed within the capsid-coding region 9 nt downstream of the initiator AUG strongly inhibited translation bothin vitroandin vivo,while multiple silent mutations within the first 42 nt of the open reading frame also reduced translation in reticulocyte lysates. Thus, domains II and III of the 5′NTR are both essential to activity of the IRES, while conservation of sequence downstream of the initiator AUG is required for optimal IRES-directed translation
Virus-like Particles and E1AE4 Protein Expressed from the Human Papillomavirus Type 11 Bicistronic E1AE4AL1 Transcript

AbstractDetection of E1AE4 protein in human papillomavirus (HPV 11)-infected tissue is tightly linked to detection of L1 major capsid protein. The only L1-containing transcript identified in HPV 11-infected tissue is the bicistronic E1AE4AL1 mRNA, potentially encoding both the E1AE4 and the L1 proteins. It has not been established that these proteins can be expressed from the E1AE4AL1 transcript. The HPV 11 E1AE4AL1 sequence was cloned by reverse transcriptase polymerase chain reaction into the p1393 vector to produce recombinant baculoviruses. Immunoblots of recombinant baculovirus-infected Sf9 cell lysates demonstrated both the E1AE4 and the L1 proteins. An ELISA was performed on infected Sf9 cells using a monoclonal antibody specific for nondenatured L1, demonstrating that 10 ng of native L1 protein was present per microgram of total nuclear protein. Electron microscopic analysis revealed 50- to 60-nm icosahedral virus-like particles.In vitrotranscription/translation was performed using pSPORT constructs containing the E1AE4AL1 sequence or, as controls, monocistronic pSPORT-E1AE4 or L1 constructs. The pSPORT-E1AE4AL1 construct produced the E1AE4 and L1 proteins at a ratio of 17:1. For E1AE4 protein, expression was greater from the pSPORT-E1AE4AL1 construct than from the monocistronic pSPORT-E1AE4 construct. In contrast, more L1 protein was expressed from pSPORT-L1 than from pSPORT-E1AE4AL1. A mutant E1AE4AL1 construct containing no E1AE4 start codon expressed L1 protein in amounts nearly equal to that expressed from the pSPORT-L1 construct. Addition of an antisense oligonucleotide directed at the E1AE4 start codon region toin vitroreactions using pSPORT-E1AE4AL1 was associated with inhibition of E1AE4 protein synthesis and increased translation of L1 protein
Characterization of Cell Lines Allowing Tightly Regulated Expression of Hepatitis C Virus Core Protein

AbstractA tetracycline-regulated system was used to generate cell lines allowing tightly controlled expression of a hepatitis C virus (HCV) cDNA comprising the 5′ noncoding, the core, and part of the E1 regions. Production of 21-kDa processed nucleocapsid protein could be regulated over a broad range by the concentration of tetracycline present in the culture medium. Induction ratios of over 1000-fold were found using an HCV core–luciferase fusion construct. Core protein had an intracellular half-life of 9 hr and corresponded to the product of 173 amino-terminal amino acids of the HCV open reading frame. Sequential immunofluorescence microscopy revealed the presence of core antigen first in a predominantly perinuclear fine-reticular staining pattern and subsequently also in cytoplasmic granules and vesicles. By immunoelectron microscopy core protein was found on the endoplasmic reticulum membrane and on the surface of cytoplasmic lipid droplets. Growth rate analyses and colony formation efficiency assays showed no major cytotoxic effect of HCV core protein expression per se. HCV gene expression could be inhibited by an antisense oligonucleotide targeting a region immediately downstream of the translation initiation codon. These cell lines represent important tools to investigate structural and functional properties of HCV core protein and may be useful to evaluate gene therapeutic strategies against HCV in a cellular system
Expression of the DNase Encoded by the BGLF5 Gene of Epstein–Barr Virus in Nasopharyngeal Carcinoma Epithelial Cells

AbstractIn contrast with most Epstein–Barr virus (EBV)-infected healthy carriers, nasopharyngeal carcinoma patients frequently have increased serum levels of antibodies directed against EBV-DNase. These antibodies are potentially interesting serological markers for the diagnosis and the follow-up of nasopharyngeal carcinoma (NPC). In this context, it is important to determine whether malignant EBV-infected cells are the source of significant amounts of EBV-DNase contributing to antigenic stimulation. Therefore EBV-DNase expression has been investigated in several NPC specimens. A significant expression of this viral enzyme was demonstrated in both fresh biopsies and transplanted tumor lines. The DNase isolated from tumor has a molecular weight varying between 52 and 60 kDa and its activity eluted from a single-stranded DNA affinity column was specifically inhibited by both NPC sera and the rabbit polyclonal antibody against EBV-DNase. The enzyme activity was functional in the presence of 300 mMKCl, with which cellular DNases are completely inhibited. The DNase was mainly localized in epithelial tumor cells of both NPC biopsies and nude mice-derived NPC cells
Complementation of a Vaccinia Virus Host-Range K1L Gene Deletion by the Nonhomologous CP77 Gene

AbstractWe investigated the host-range restriction of a vaccinia virus (VV) K1L deletion mutant in rabbit kidney RK13 cells and the ability of the nonhomologous cowpox virus CP77 gene to overcome this block. Viral early mRNAs were made by K1L−VV but early protein synthesis was arrested consistent with a translational block. Replication of viral DNA did not occur and neither intermediate nor late mRNAs or proteins were detected. These results indicated that host-range restriction occurs earlier in RK13 cells than in Chinese hamster ovary cells (CHO) cells infected with CP77−VV, where the block occurs at translation of intermediate stage mRNA. We confirmed a report (Perkuset al., Virology179, 276–286, 1990) that the CP77 gene, which allows VV replication in CHO cells, could replace the K1L gene for plaque formation in RK13 cells. However, the size of the plaques formed by K1L−CP77+VV was smaller than those formed by K1L+CP77−VV. Single-step growth curves also showed that the CP77 gene could functionally replace the K1L gene, although formation of infectious virus was delayed and did not reach the same level as that of K1L+VV. Most surprisingly, the dramatic shutoff of viral and host gene expression was similar in RK13 cells infected with K1L−CP77−VV and K1L−CP77+VV and little difference was noted for the first 6 hr. Subsequently, in cells infected with the K1L−CP77+VV, viral early protein synthesis was spontaneously resurrected and the replication cycle proceeded. Despite the absence of homology, K1L and CP77 gene products appear to be acting in a common virus/cell interaction pathway
Syngeneic Marek's Disease Virus (MDV)-Specific Cell-Mediated Immune Responses against Immediate Early, Late, and Unique MDV Proteins

AbstractMarek's disease (MD) infection has been controlled effectively by vaccination using nononcogenic and/or attenuated oncogenic Marek's disease virus (MDV) vaccines. Thus far, there is little knowledge on the role of cell-mediated immune (CMI) responses during MDV infection or vaccination. To elucidate the importance of MDV proteins in CMI responses, the pp38, Meq, ICP4, or ICP22 genes of an oncogenic strain, GA and the gB, ORF A, A41, or L1 genes of a highly oncogenic strain, RB1B were stably transfected into reticuloendotheliosis virus (REV)-transformed lymphoblastoid cells, CU-91 (MHC:B19B19) and CU-205 (MHC:B21B21). Cell lines positive for MDV gene transcription and/or protein expression were used in a standard 4-hr chromium release assay. Effector cells for this assay were obtained from splenocytes of chickens infected with the oncogenic strain, JM-16/13 or the nononcogenic vaccine strain, SB-1/12. Cell lines expressing MDV pp38, Meq, or gB were lysed by syngeneic but not allogeneic MDV-sensitized splenocytes obtained from chickens ofB19B19andB21B21haplotypes. However, syngeneic CMI responses against ICP4 were detected only inB21B21chickens. CMI responses were not detected againstB19B19andB21B21cell lines expressing A41, L1, ORF A, or ICP22. This report suggests that syngeneic CMI responses against pp38, Meq, ICP4, and gB of GA and RB1B strains, respectively, can be induced in chickens inoculated with JM16/13 or SB-1/12. The difference in CMI response to ICP4 in genetically susceptible (B19B19) and genetically resistant (B21B21) chickens may be an important factor in genetic resistance