pinecone/core-2020-05-10-deduplication huggingface dataset:
Development of a Sustainable Chick Cell Line Infected with Marek's Disease Virus

AbstractMarek's disease virus (MDV) is a highly infectious and cell-associated avian herpesvirus. Fully productive infections with MDV are restricted to feather follicle epithelium of afflicted birds. In culture, MDV infection of primary chick (CEF) and duck embryo fibroblast cells is semiproductive. Passage of MDV and production of MDV vaccines is limited to these primary cell-associated systems. The finite life span of primary avian cell cultures has hampered efforts to use positive selection in generation of recombinant MDV and complicates studies of temporal gene regulation. In this report, we describe continuous chick fibroblast cell lines (MDV OU2.2 and MDV OU2.1) which support MDV replication. Southern blot and PCR analyses demonstrate that these cell lines harbor MDV DNA. Western blot analyses indicate that MDV OU2.2 cells express at least a limited set of viral proteins, pp38 and pp14, similar to that seen in MDV-lymphoblastoid cells. Presence of distinct plaques in confluent MDV OU2.2 cell monolayers is consistent with cytolytic semiproductive infection, similar to that observed in primary CEF. MDV OU2.2 cells are capable of transferring MDV infection to primary CEF cultures and inducing clinical signs of Marek's disease in susceptible birds. MDV OU2.2 cells have maintained a MDV-positive phenotype for over 16 months of active culture. Southern blot hybridization of MDV OU2.2 cell DNA reveals a distinct expansion of the MDVBamHI H fragment in a subset of viral genomes following long-term cultivation
Canyon Rim Residues, Including Antigenic Determinants, Modulate Serotype-Specific Binding of Polioviruses to Mutants of the Poliovirus Receptor

AbstractSeveral mouse cell lines expressing hybrid human poliovirus receptors (hPVRs) bearing mutations in the first immunoglobulin-like domain were previously characterized for their defective binding and replication of poliovirus type 1 Mahoney (G. Bernhardt, J. Harber, A. Zibert, M. DeCrombrugghe, and E. Wimmer,Virology,203, 344–356, 1994). Here we report that these mutant hPVRs were utilized to explore differences in the binding behavior of the three serotypes of poliovirus. Type 3 polioviruses (both Sabin and the neurovirulent Leon strain) clearly bound to the hPVR mutant Q130G/GD, but were incapable of initiating infection. Also, binding at 25° of poliovirus types 2 and 3 to cell lines expressing the hPVR mutants P84SYS/HPGA, L99GAE/AAAA, and D117F was greater than type 1 poliovirus. Further study of the serotype-specific interaction with mutant hPVRs was accomplished with antigenic hybrid viruses. Improved binding by antigenic hybrid viruses demonstrated that serotype-specific binding to mutant hPVRs is, in part, determined by the amino acid sequence of neutralization antigenic sites (NAgs) and the probable conformational rearrangement of amino acids adjacent to the NAg sites. Finally, site-directed mutants of poliovirus were utilized to determine the relative contributions, to hPVR interactions, of individual amino acids with solvent accessible side chains in the viral canyon. Of the 18 viable virus mutants produced, 3 (D1226A, I1089A, and VPEK1166HPGA) expressed impaired replication phenotypes on the mutant hPVR cell lines P84SYS/HYSA and D117F. A location at the rim of the poliovirus canyon was implicated for the interaction of the amino terminal domain of the poliovirus receptor with conserved and serotype-specific viral surface amino acids. The possible involvement of elements of neutralization antigenic sites in receptor binding may explain, in part, why poliovirus exists in only three serotypes
Structural Comparison of the Plant Satellite Viruses

AbstractDetailed structures are now available for three plant satellite viruses, satellite tobacco necrosis virus (STNV), satellite tobacco mosaic virus (STMV), and satellite panicum mosaic virus (SPMV). It is, therefore, possible to compare the tertiary structure of viral protein subunits, their quaternary interactions, and the interactions of protein subunits with the RNA genome. This analysis indicates that, in spite of common function and preservation of a “jelly-roll” motif in the protein monomer, the three viruses are remarkably different. The differences include the arrangement of secondary structural elements, interactions of adjacent subunits, and the disposition of subunits relative to icosahedral symmetry axes. In each of the three viruses, however, the narrow end of the jelly roll forms fivefold contacts. The fivefold protein interactions are organized about a Ca2+ion for STNV, an anion for STMV, and, apparently, neither of these for SPMV. Low-resolution neutron diffraction studies using H2O/D2O solvent contrast variation revealed the general location of the RNA genome within the STNV. In the case of SPMV, regions of electron density on the interior of the capsid could be assigned to RNA, although it was not possible to model the nucleic acid. Only for STMV was nucleic acid visible in election density maps, and this was manifested as double-helical RNA segments associated with each coat protein dimer. The observations presented here provide no support for any common evolutionary relationship
Feline Leukemia Virus Variants in Experimentally Induced Thymic Lymphosarcomas

AbstractThis study was initiated to evaluate thein vivoinfectivity and pathogenicity of a group of recombinant feline leukemia viruses (rFeLVs) previously generated byin vitroforced recombination between a FeLV subgroup A virus (FeLV-A) and an endogenous FeLV (enFeLV) envelope (env) element (Sheetset al.,1992,Virology190, 849–855). To determine infectivity of rFeLVs, neonatal cats were inoculated with rFeLVs alone or in combination with FeLV-A. The recombinant viruses were able to replicate efficientlyin vivoonly when administered along with FeLV-A. Of six co-infected cats, three developed thymic lymphosarcomas, one severe aplastic anemia, and two cachexia and depression; all were viremic and seroconverted shortly after inoculation. While both virus types were detected in virtually all tissues examined from these tumor-bearing cats, there was a particularly noteworthy sequence reversion in the rFeLVs. It is known that exogenous FeLV isolates carry a conserved neutralizing MGPNL epitope in the middle of the surface glycoprotein domain of theenvgene. In contrast, the parental recombinant viruses used to inoculate these cats harbored the enFeLV-derived MGPNP sequence at this position. However, allin vivo-propagated recombinants displayed the MGPNL sequence, while theenv-encoded backbone flanking the MGPNL sequence was that of the parental recombinant virus. These results suggest that viruses with the MGPNL epitope have anin vivoproliferative advantage. The data also provide an explanation for the conservation of this epitope in exogenous FeLVs despite the existence of variant forms in enFeLV proviral elements with which they can recombine
Synthesis of Bluetongue Virus Chimeric VP3 Molecules and Their Interactions with VP7 Protein to Assemble into Virus Core-like Particles

AbstractBluetongue virus (BTV) core-like particles (CLPs) are formed in the cytoplasm of insect cells when only two major proteins (VP3 and VP7) of the BTV core are expressed by baculovirus vectors (T. J. French and P. Roy, 1990,J. Virol.64, 1530–1536). We have recently reported that five small internal deletion mutants of VP3 form CLPs when provided with unmodified VP7 protein (D1–5; S. Tanaka and P. Roy, 1994,J. Virol.68, 2795–2802). To investigate whether foreign sequences can be inserted into VP3 and to determine their effect on CLP formation, three of these internal regions (D1, D2, and D5), as well as the carboxy terminus, were modified to create unique restriction enzyme sites, thereby replacing VP3 coding regions with shorter synthetic sequences. Each modified VP3 gene was used to generate baculovirus expression vectors (D1I, D2I, D5I, and VP3C, respectively). Other than the D5I mutant, the mutants formed CLPs when expressed in the presence of VP7. Subsequently, T7 tag epitopes were inserted into the D1I, D2I, and VP3C restriction sites and recombinant baculoviruses were generated to express chimeric VP3 proteins (VP3D1IT, VP3D2IT, and VP3CT). Each chimeric protein retained the ability to form CLPs when VP7 protein was provided. In another construction an immunogenic sequence representing a bovine leukemia virus (BLV) glycoprotein peptide was incorporated into the carboxy terminus of VP3 and the derived CLPs were used to raise antibodies that reacted with the BLV antigen. The results suggest that the VP3 molecule can accommodate foreign sequences at certain sites without perturbing their ability to form CLPs with VP7
Naturally Occurring Sin Nombre Virus Genetic Reassortants

AbstractGenetic reassortment has been shown to play an important role in the evolution of several segmented RNA viruses and in the epidemiology of associated diseases. Sin Nombre (SN) virus is the cause of hantavirus pulmonary syndrome throughout the western United States. Like other hantaviruses, it possesses a genome consisting of three negative-sense RNA segments, S, M, and L. Recent analysis has demonstrated the presence of at least three different hantaviruses in Nevada and eastern California, including SN, Prospect Hill-like, and El Moro Canyon-like viruses. In addition, two distinct lineages of SN virus can be found inPeromyscus maniculatusrodents (sometimes in close proximity) trapped at study sites in this region. Data obtained by phylogenetic analysis of sequence differences detected among the S, M, and L genome segments of these SN viruses are consistent with reassortment having taken place between SN virus genetic variants. The results suggest that M (and to a lesser extent S or L) genome segment flow occurs within SN virus populations inP. maniculatusin this region. No reassortment was detected between SN virus and other hantavirus types present in the area. This finding suggests that as genetic distance increases, the frequency of formation of viable reassortants decreases, or that hantaviruses which are primarily maintained in different rodent hosts rarely have the opportunity to genetically interact
Infectious Transcripts of Tick-Borne Encephalitis Virus, Generated in Days by RT-PCR

AbstractConstruction of infectious clones of flaviviruses can be problematic owing to instability, toxicity, and recombination events occurring while cloning cDNA in the bacterial vectors. To overcome these difficulties we have devised a rapid and simple method for producing an infectious genetically engineered tick-borne encephalitis virus in less than 10 days using viral RNA from an unpurified virus suspension. The experimental protocol utilized the high fidelity reverse transcription-polymerase chain reaction to produce two long (5.7 and 5.2 kb) overlapping cDNA segments. To produce full-length cDNA the two overlapping segments were either ligated or fused by polymerase chain reaction. The cDNA was then transcribed and the derived full-length RNA was injected intracerebrally into young mice which reproduced the infectious virus within 8–20 days. To differentiate the engineered virus from parent virus, aSunI restriction site was introduced by substituting nucleotides at positions 5688 and 5691 of the viral genome. This restriction site was present in the engineered virus recovered from infected mice. Antigenic and electrophoretic analysis of the proteins recovered from the engineered virus confirmed that it was indistinguishable from parent virus. In addition to its applicability as a rapid method of producing infectious engineered virus, this protocol offers the opportunity to introduce changes by site-directed mutagenesis without needing to clone the viral DNA. The method should be applicable to most viruses possessing an infectious RNA molecule and reduces the time required to produce a genetically engineered virus from years to days. When appropriate, the choice of mice for transfection of RNA has the advantage of being extremely simple, very sensitive, and producing high titers of stable virus
In VivoProcessing of Pr160gag-polfrom Human Immunodeficiency Virus Type 1 (HIV) in Acutely Infected, Cultured Human T-Lymphocytes

AbstractThe processing of the HIV-1 Pr160gag-polprecursor polyprotein was analyzed in freshly HIV-1-infected MT-4 cultured cells. Single intermediates of the processing cascade were characterized by immunoblotting using distinct antisera. A potent inhibitor of the HIV protease (PR), Ro 31-8959, was employed to block cleavage by the mature PR, thus allowing insights into initial stages of thegag-pol(auto)-catalytical processing. While most knowngag-polcleavages were blocked in the presence of the inhibitor, the cleavage site between thegag-NC and thepol-p6* domains was still cleaved even in presence of high amounts (1 μM) of inhibitor, leading to the accumulation of a novel 114-kDa polyprotein comprising p6*-PR-RT-IN. In the absence of inhibitor no accumulation of p114 was observed. In inhibitor-treated, HIV-1-infected cells a p6*-PR intermediate was also detected, indicating subsequent cleavage of the PR/RT scissile bond. These results demonstrate initial cleavage(s) of thegag-polprecursor hydrolyzed by a proteolytic activity different from the mature PR and indicate that p114 (p6*-PR-RT-IN) and p6*-PR intermediates could play an essential role in the PR activation process
Isolation of Cytotoxic T Lymphocytes from Healthy Seropositive Individuals Specific for Peptide Epitopes from Epstein–Barr Virus Nuclear Antigen 1: Implications for Viral Persistence and Tumor Surveillance1

AbstractThe question of whether Epstein–Barr nuclear antigen 1 (EBNA1) includes cytotoxic T lymphocyte (CTL) epitopes has generated considerable scientific interest, primarily due to its important implications for the overall biology of Epstein–Barr virus (EBV). Earlier studies have suggested that EBV-associated malignancies that express only EBNA1 escape virus-specific immune surveillance since this antigen is not a target for CTL recognition. In the present report we have used a modified protocol to demonstrate that EBNA1 includes sequences which can be recognized by both polyclonal and clonal CTLs. CD4+CTL clones were isolated from a healthy, seropositive donor that recognized the peptide epitope TSLYNLRRGTALA from EBNA1 in association with HLA DR1. Interestingly, these CTLs are unable to lyse EBV-infected B cells suggesting that EBNA1 may not be endogenously processed and/or presented to the host CTL response. Despite recent suggestions that glycine–alanine repeat sequences within EBNA1 can inhibit endogenous processing, target cells infected with recombinant vaccinia vectors encoding truncated EBNA1 proteins, without these repeat sequences, were not recognized by this CTL clone. Thus it seems that the presence of glycine–alanine repeats is not responsible for inhibiting the processing of the class II-restricted epitope defined in this study. These results substantiate the view that EBV-infected normal and malignant cells, where viral gene expression is limited to EBNA1, can resist CTL-mediated immune surveillancein vivo
Interaction of Host Proteins with the Plus-Strand Promoter of Brome Mosaic Virus RNA-2

AbstractThe binding of five barley proteins (Mr: 37, 36, 35, 34, and 30 kDa) to the ICR2 motif present at the 5′ end of brome mosaic virus (BMV) RNA-2 was identified using UV cross-linking. Evidence that the interaction is specific included the observation that these proteins did not recognize a similar-size RNA fragment transcribed from a nonviral (β-glucuronidase) gene, nor did they bind to the 3′ end of the plus strand of RNA-3. Replication-defective BMV RNA-2 mutants bearing substitution mutations at nucleotides 9 and 10 of the ICR2 motif were used to show that these positions were involved in the interaction of the five barley proteins with BMV RNA-2. Surprisingly, the profile of barley proteins interacting with the 3′ end of the minus strand of RNA-2 was similar to that seen for the 5′ end of the plus strand. Further, the profile of proteins binding to minus-sense probes bearing substitution mutations in the ICR2 region differed from that found for the wild-type sequence. These findings support the concept that host proteins are involved in genome replication and that their ability to interact with both plus and minus strands of the viral RNA is probably involved with the initiation of plus-strand synthesis
Molecular Basis for the Resistance of Influenza Viruses to 4-Guanidino-Neu5Ac2en

AbstractWe report the selection and characterization of influenza A/NWS-G70c and B/HK/8/73 (HG) viruses which are resistant to the potent influenza neuraminidase inhibitor, 4-guanidino-Neu5Ac2en. Viruses were selected which replicated in MDCK cells in the presence of 20 μg/ml inhibitor. The neuraminidase of resistant viruses was >200-fold more resistant to 4-guanidino-Neu5Ac2en than was the neuraminidase of the parent viruses. Although amounts of neuraminidase protein were similar in resistant and parent viruses, the enzyme activity of the resistant neuraminidase heads was reduced by >95% for the substrates used. Relative to parent viruses, the resistant viruses replicated to equal or greater titers in tissue culture and in embryonated chicken eggs. Sequence analysis revealed a single nucleotide mutation in the neuraminidase gene of each virus resulting in the change of the conserved Glu 119 (which lies in a pocket beneath the active site of the enzyme) to Gly thus eliminating an electrostatic interaction with the C-4 guanidinium moiety of the inhibitor. Mutations (Asn → Ser) at amino acids 145 and 150 were also found in the hemagglutinin gene of the B/HK/8/73 (HG) virus resistant to 4-guanidino-Neu5Ac2en. No changes were found in the hemagglutinin gene of the resistant A/NWS-G70c virus
The Carboxy-Terminal Domain Is Essential for Stability and Not for Virion Incorporation of HIV-1 Vpr into Virus Particles

AbstractVpr is one of the auxiliary gene products encoded by HIV-1 genome. Vpr is a 14-kDa protein and exhibits several interesting characteristics including incorporation into virus particles, oligomerization, localization in the nucleus, and positive regulation of virus replication in primary cells. In an effort to define the structure–function relationship of Vpr, the role of the C-terminus of Vpr was investigated. Site-specific mutagenesis involving deletion, insertion, and substitution of residues at the C-terminus was utilized to generate variants of Vpr. Mutations introduced at the C-terminus affected properties of Vpr in different ways: (i) Vpr containing amino acids 1–72 showed the virion incorporation phenotype, indicating that the C-terminus is not essential for this function, (ii) the C-terminus contributes to the stability of Vpr, and (iii) substitution mutagenesis involving the basic residues showed stability similar to that of wild type, indicating the lack of involvement of these residues in this biochemical property of Vpr. The data generated in this study and our early mutagenic analyses on Vpr suggest that domains noncontiguous in primary sequence contribute to the stability of Vpr through overall conformation of the protein
Human PKR Transfected into Murine Cells Stimulates Expression of Genes under Control of the HIV1 or HTLV-I LTR

AbstractWe have analyzed the effect of transfection into murine NIH/3T3 cells of the human dsRNA-activated kinase PKR on the expression of the β-galactosidase reporter gene, placed under control of the HIV1 or the HTLV-I LTR. β-Galactosidase expression is stimulated when the reporter plasmids are cotransfected with wild-type PKR but inhibited when cotransfected with a catalytically inactive mutant PKR. In the case of HIV1, β-galactosidase expression was not stimulated when cotransfection was carried out with PKR harboring mutations in the dsRNA binding domains, indicating that stimulation depends on the classical mode of PKR activation through dsRNA binding. In contrast, the dsRNA binding mutants of PKR could still partially stimulate β-galactosidase expression from the HTLV-I LTR, suggesting that PKR activation in this case may involve different/additional mechanisms. These results show that, in addition to the known down-regulation of protein synthesis through eIF2 phosphorylation, PKR can also positively stimulate gene expressionin vivo,most probably through phosphorylation of a substrate distinct from eIF2
cis-Acting Elements of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

AbstractTranslation initiation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal entry into the 5′ untranslated region. Internal ribosome binding to EMCV mRNA requires a viralcis-acting element, termed the internal ribosomal entry site (IRES), and cellulartrans-acting factors. The polypyrimidine tract binding protein (PTB) has been identified as one suchtrans-acting factor required for EMCV IRES-dependent translation. Using a dicistronic mRNA and anin vitrotranslation system, we have identifiedcis-acting elements of the EMCV IRES required for IRES-dependent translation. The results identify several regions of the IRES that are required for efficient IRES-dependent translation, including the PTB binding site. Other regions of the IRES may act only as spacer sequences, analogous to the spacer sequences found in rhinovirus and enterovirus IRES elements, to link essential regions of the IRES together. The flexibility of one region of the EMCV IRES was demonstrated by an insertion of 125 nucleotides that had little effect on IRES function while the constraint imposed on another region was demonstrated by a 3-nucleotide deletion that nearly abolished IRES-dependent translation
Sequence Variation in the Capsid Protein Genes of Human Papillomavirus Type 16 and Type 31

AbstractThe sequences of the capsid genes of a human papillomavirus type 16 (HPV 16) DNA and an HPV 31 DNA were determined. The HPV 16 DNA contained genes coding for the most variable HPV 16 capsid proteins yet identified (17 variable amino acids). Three of six coding changes in the HPV 31 DNA occurred at positions equivalent to ones where variable amino acids in HPV 16 have been observed. Variable amino acids in both viruses occurred predominantly in regions which showed amino acid variation when closely related types of HPV were compared; thus, most of the factors which determined the intratypic variation in the capsid proteins of the viruses described here were likely the same as those which determined differences between the capsid proteins of different HPV types
The African Swine Fever Virus IAP Homolog Is a Late Structural Polypeptide

AbstractThe analysis of the complete nucleotide sequence of the African swine fever virus genome has revealed the existence of a number of genes potentially capable of modifying the host's response to the virus infection. In this report, we describe the results of the characterization of the A224L gene that encodes a novel member of the family of apoptosis inhibitors known as IAP proteins. A224L is expressed during the late phase of the infectious cycle, and its polypeptide product is assembled into virus particles
Epstein–Barr Virus Nuclear Antigen 2–Estrogen Receptor Fusion Proteins Transactivate Viral and Cellular Genes and Interact with RBP-Jκ in a Conditional Fashion

AbstractEpstein–Barr virus nuclear antigen 2 (EBNA2) is a transcriptional activator of viral and cellular genes involved in B cell transformation by EBV and is targeted to EBV responsive promoters through interaction with cellular DNA binding proteins such as RBP-Jκ. To develop a conditional system in which the function of EBNA2 can be switched on and off, we have fused the hormone binding domain of the estrogen receptor to the N- or C-terminus of EBNA2. Here we show that after transient or stable transfer of these chimerical EBNA2 genes into human B cell lymphoma lines, transactivation of LMP1, TP1, and TP2 promoter constructs, expression of the cell surface markers CD21 and CD23, and binding of EBNA2 to its cellular partner RBP-Jκ are dependent on the presence of estrogen. The EBNA2 fusion proteins proved to be virtually inactive in the absence of hormone