pinecone/core-2020-05-10-deduplication huggingface dataset:
Bacteriophage Mu Head Assembly

AbstractThe protein composition of defective particles produced by various bacteriophage Mu head-gene mutants was analyzed by SDS–PAGE. An abundant 20-kDa protein was detected in only one type of defective head. This protein exhibits properties of a scaffolding protein. A 50-kDa structural protein present in most defective heads was shown to be produced by cleavage of the C-terminus of the 64-kDa polypeptide encoded by geneH.Cleavage occurs during head assembly at a site which, according to earlier results, might separate two different functional domains in gpH.A fraction of the gpHmolecules produced upon Mu induction sediment in a 25 S complex, suggesting that gpHparticipates in the formation of an early intermediate of Mu head assembly. Characteristics of gpHsuggest that it may be the Mu portal protein
HIV-1 Infection of Cultured Human Adult Oligodendrocytes

AbstractThe mechanism through which HIV-1 causes HIV dementia (HIVD) is not well understood. Myelin pallor is a common pathological finding in HIVD and could be explained by a direct infection of oligodendrocytes or interaction with HIV-1 gp 120. To determine if oligodendrocytes could be infected by HIV-1, we purified oligodendrocytes from adult human brain tissue obtained from temporal lobe resections. These cells were exposed to HIV-1 and infectivity was assayed by detection of p24gagantigen, PCR amplification, and cocultivation with CD4+cells. These results indicate that HIV-1IIIB and BaLand one of four primary isolates tested can infect oligodendrocytes, resulting in the production of infectious virus. Furthermore, in an experiment that mimics a potentialin vivoscenario, infected microglia were able to transmit virus to oligodendrocytes in a trans-well culture system. These experiments indicate that oligodendrocyte infection should be considered in studying the pathophysiology of HIVD
Persistence of Japanese Encephalitis Virus Is Associated with Abnormal Expression of the Nonstructural Protein NS1 in Host Cells

AbstractPersistent infection with Japanese encephalitis virus (JEV) was established in murine neuroblastoma N18 cells, and the persistency has been maintained in cell culture for over 6 months. From the persistently infected cells, a clone named C2-2 was selected and expanded to form a stable cell line. The vast majority of C2-2 cells showed viral protein staining by immunofluorescence and continuously produced low levels of virus (103to 104PFU/ml) without marked cytopathic effects or cyclic variations. In addition to the wild-type viral proteins, truncated forms of the viral nonstructural protein 1 (NS1) as well as its derivative NS1′ were produced in C2-2 cells. Both truncated NS1 and NS1′ contain deletions at their N-termini; however, the analyses by RT–PCR and direct sequencing of the viral RNA failed to detect any truncations or mutations within the NS1 region, suggesting that NS1 truncation was a result of a unique posttranslational proteolytic cleavage of NS1 in the persistently infected cells. Similar but not identical truncation of NS1 was also observed in two other persistently infected cell lines established in Vero and DBT (murine astrocytoma) cells. However, viruses released from C2-2 cells did not produce truncated NS1 upon infection of N18 cells, suggesting that NS1 truncations were the result of virus–cell interaction in persistently infected cells. These data indicate a strong association between abnormal NS1 expression and JEV persistency. A probable involvement of dysfunctional NS1 in the establishment and/or maintenance of JEV persistency in tissue culture is discussed
Bicistronic and Two-Gene Retroviral Vectors for UsingMDR1 as a Selectable Marker and a Therapeutic Gene

AbstractWe describe a series of two-gene and bicistronic retroviral vectors that use the humanMDR1 gene as a selectable marker for the overexpression of a second heterologous gene in transduced cells. The vectors use Harvey murine sarcoma virus sequences for viral expression and packaging functions and include sites for cloning foreign genes of interest under the control of either an internal promoter (two-gene vectors) or an internal ribosome entry site (bicistronic vectors). To characterize these vectors, we usedneoas a reporter gene for foreign gene expression and as an independently selectable marker for comparison withMDR1. Each of the vector constructions supported high-titer retrovirus production and transduction of mouse and human cell lines. UsingMDR1–neovirus supernatants in parallel titering assays, we found that titers based on colchicine resistance were 10- to 20-fold lower than titers based on G418 resistance, suggesting thatMDR1 is a more stringent selectable marker thanneoin NIH 3T3 and KB-3-1 cell lines. Whereasneogene expression with the two-gene vectors was subject to host-specific limitations on internal promoter activity, the bicistronic vectors were highly active in three cell lines tested. In K562 cells, using the bicistronic vector, selection with colchicine led to at least 20-fold higher expression of theMDR1 gene product than did selection with G418, suggesting that the stringentMDR1 selection system is very efficient for obtaining overexpression of foreign genes. Retroviral vectors carryingMDR1 as a selectable marker plus a second, heterologous gene of interest could have widespread utility forin vitroandin vivoapplications of gene transfer technology, including gene therapy
Nonconserved Nucleotides at the 3′ and 5′ Ends of an Influenza A Virus RNA Play an Important Role in Viral RNA Replication

AbstractThe genome of influenza A viruses is composed of eight negative-strand RNA segments which contain short noncoding regions at their 3′ and 5′ ends. The signals required for replication, transcription, and packaging of the viral RNAs are thought to be located in these regions. The highly conserved noncoding nucleotides, which form “panhandle” or “fork” structures by partial complementarity, are important for the transcriptional activity of the viral RNA polymerase. In contrast, the nonconserved noncoding nucleotides located close to the open reading frame of the viral RNAs had not been implicated in RNA transcription. Using a reverse-genetics system, we have now rescued influenza A/WSN/33 viruses whose NA-specific RNA segments have deletions in these nonconserved noncoding regions. Deletion either of the nucleotide residues between the poly(U) stretch and the stop codon at the 5′ end or of the nucleotides between position 15 and the start codon at the 3′ end did not affect the amount of NA-RNA species found in virions or infected cells. However, a combination of deletions at both the 3′ and the 5′ ends decreased by 60 times the levels of NA-specific viral RNA found in infected cells at late periods of infection and in virions. This double deletion was also responsible for a fourfold reduction of the steady-state levels of the NA-specific mRNA in infected cells. Viruses whose NA-specific open reading frames were flanked by the noncoding regions of the PB1- or the NS-RNA segments of influenza A/WSN/33 virus also showed a reduction in the NA-specific viral RNA in virions and in infected cells. The present results demonstrate that the nonconserved nucleotides at the 3′ and 5′ ends of the NA-RNA segment of influenza A virus play an important role in the replication of this segment
Antigenic Mapping of the Recombinant Norwalk Virus Capsid Protein Using Monoclonal Antibodies

AbstractNorwalk virus (NV) is the prototype strain of a group of noncultivatable caliciviruses that infect humans and cause outbreaks of epidemic acute nonbacterial gastroenteritis. The NV virion is composed of 180 copies of a single structural protein that, when expressed in insect cells infected with a recombinant baculovirus, assembles into empty recombinant Norwalk virus-like particles (rNV VLPs) which are morphologically and antigenically similar to native NV. We have begun to dissect the antigenic structure of the rNV particles using monoclonal antibodies made to the rNV VLPs. Ten MAbs made to rNV particles were characterized for their reactivity as detector antibodies by ELISA, as capture antibodies in an ELISA to detect NV in stools, by Western blot, and by immunoprecipitation. Seven of the MAbs recognize discontinuous epitopes, requiring the rNV capsid protein to remain at least partially folded, while the other three recognize continuous epitopes. Eight of the MAbs map to the C-terminal half of the capsid protein as they react by Western blot and by immunoprecipitation with a 32K trypsin cleavage product of the full-length 58K capsid protein, suggesting that the C-terminal half of the capsid protein may contain the immunodominant epitopes. The three MAbs that recognize continuous epitopes map to the extreme C terminus of the capsid protein, between amino acids 457 and 530, in a region that is relatively conserved among different human calicivirus capsid proteins. These MAbs which were assigned into three antigenic groups will be useful as tools to further dissect the structural and antigenic topography of the NV virion, and as unlimited reagents to detect NV in diagnostic assays
Bacterial Expression of Neutralizing Mouse Monoclonal Antibody Fab Fragments to Hantaan Virus

AbstractWe amplified by polymerase chain reaction the heavy and light chain antibody genes of two mouse hybridomas secreting neutralizing monoclonal antibodies (MAbs) to the G1 or G2 envelope proteins of Hantaan virus, cloned them into the phagemid vector pComb3, and expressed them in bacteria to yield Fab fragments. Expressed Fab fragments had the same antigenic specificities for Hantaan and Seoul viruses as the complete parent MAbs and were able to neutralize Hantaan virus in plaque-reduction neutralization assays. The authentic MAb to G2 (HCO2) could passively protect hamsters from challenge with Hantaan virus when neutralizing antibody titers of at least 1:10 were detected in the animals’ sera just prior to challenge. In contrast, although 1:10 neutralization titers were also detected in hamsters receiving passively transferred,Escherichia coli-expressed HCO2 Fab, these animals were not protected from infection with Hantaan virus. Similarly, passive transfer of the HCO2 MAb on Days 1 through 4 after infection prevented antigen deposition in hamster lungs and kidneys but passive transfer of the recombinant HCO2 Fab did not. The results suggest that although neutralization by IgG antibodies correlates with protection in hamsters, the same may not be true for neutralizing Fab fragments
Physical and Functional Characterization of the Double-Stranded RNA Binding Protein Encoded by the Vaccinia Virus E3 Gene

AbstractThe vaccinia virus E3 gene encodes a 190-amino acid double-stranded (ds) RNA-binding protein that antagonizes cellular antiviral response pathways triggered by dsRNA and interferon. The physical and functional properties of the E3 protein were determined using recombinant E3 produced in bacteria and purified to homogeneity. We show by sedimentation and chemical crosslinking that E3 is a dimer in solution at high ionic strength. E3 self-associates to form higher order oligomers as ionic strength is reduced from 1 to 0.1MNaCl. Structure probing by limited proteolysis suggests that E3 consists of amino- and carboxyl-terminal domains separated by a trypsin-sensitive bridge at residues Lys-92 and Arg-95. The carboxyl-domain of E3 contains a conserved dsRNA binding motif (dsRBM) found in many other proteins that interact with dsRNA. That the C-terminal domain per se binds to dsRNA was verified by studies of recombinant E3(100–190) purified from bacteria. The affinity of the C-terminal domain for dsRNA was comparable to that of the full-length E3 protein (KD∼ 7 to 9 nM). E3(100–190) did not bind to DNA–DNA duplexes or to DNA–RNA hybrids, suggesting that the dsRBM specifically recognizes an A-form helix. E3(100–190) is a dimer in solution; however, unlike the full-sized E3 protein, E3(100–190) does not form higher order multimers at low ionic strength
Sensitivity of p53 Lysine Mutants to Ubiquitin-Directed Degradation Targeted by Human Papillomavirus E6

AbstractThe activity of the p53 tumor suppressor protein is regulated, at least in part, through the stability of the protein. p53 degradation in normal cells is controlled by ubiquitin-dependent proteolysis, and activation of p53 following DNA damage is associated with an increase in the stability of the protein. The human papillomavirus-encoded E6 protein abrogates p53 function by targeting it for rapid degradation, also through the ubiquitin pathway. Although the p53 protein is ubiquitinated following interaction with E6, we show here that none of the lysine residues within p53 are specifically required for E6-targeted degradation. Mutation of lysine residues within the C-terminus of p53 resulted in resistance to E6-mediated degradationin vitro,although the ability of the two proteins to form a complex was not affected. The same mutant was efficiently targeted for degradation in cells, however, illustrating a lack of correlation between thein vitroand thein vivoassays
Mutational Analysis of HIV-1 Nef: Identification of Two Mutants That Are Temperature-Sensitive for CD4 Downregulation

AbstractThe Nef protein of HIV-1 is capable of performing at least two functions: reduction of cell-surface expression of CD4, the viral receptor, and enhancement of HIV-1 infectivity. Here we report the results of assays of CD4 downregulation bynefpoint mutants. Areas of high charge density and highly conserved sequence motifs were targeted. In general, mutations in the carboxyl-terminal half of the protein had more deleterious effects than did those in the amino-terminal half. A single mutantnefallele was identified which encoded a stable protein but was completely inactive. Two mutant Nef proteins exhibited temperature-sensitive phenotypes in assays of CD4 downregulation
The BPV-1 E2 DNA-Contact Helix Cysteine Is Required for Transcriptional Activation but Not Replication in Mammalian Cells

AbstractThe papillomavirus E2 protein contains an amino-terminal region thought necessary and sufficient to support transcriptional activation and a carboxy-terminal region shown to direct sequence-specific DNA binding and dimerization. A cysteine residue in the center of the E2 DNA recognition helix is highly conserved among papillomavirus E2 proteins. Mutations of this cysteine in bovine papillomavirus type 1 E2 to serine and glycine resulted in proteins which failed to activate E2-dependent promoters in mammalian cells. These E2 mutants were DNA-binding competent, dimeric, and nuclear. When fused to the VP16 transactivation domain, C-terminal regions of E2 containing the mutations at 340 supported transcriptional activation, indicating that the heterologoustrans-activation domain did not require cysteine in the DNA-binding helix as did the full-length E2 transactivating protein. Although cysteine-340 was required for transcriptional activation it was not required for DNA replicationin vivo.Together, these results suggest that the E2 DNA-binding domain may directly contribute to functions of transcriptional activation previously thought limited to the N-terminal domain
Coronavirus Transcription Mediated by Sequences Flanking the Transcription Consensus Sequence

AbstractIn our studies of murine coronavirus transcription, we continue to use defective interfering (DI) RNAs of mouse hepatitis virus (MHV) in which we insert a transcription consensus sequence in order to mimic subgenomic RNA synthesis from the nondefective genome. Using our subgenomic DI system, we have studied the effects of sequences flanking the MHV transcription consensus sequence on subgenomic RNA transcription. We obtained the following results. (i) Insertion of a 12-nucleotide-long sequence including the UCUAAAC transcription consensus sequence at different locations of the DI RNA resulted in different efficiencies of subgenomic DI RNA synthesis. (ii) Differences in the amount of subgenomic DI RNA were defined by the sequences that flanked the 12-nucleotide-long sequence and were not affected by the location of the 12-nucleotide-long sequence on the DI RNA. (iii) Naturally occurring flanking sequences of intergenic sequences at gene 6–7, but not at genes 1–2 and 2–3, contained a transcription suppressive element(s). (iv) Each of three naturally occurring flanking sequences of an MHV genomic cryptic transcription consensus sequence from MHV gene 1 also contained a transcription suppressive element(s). These data showed that sequences flanking the transcription consensus sequence affected MHV transcription
A New Form of Particulate Single and Multiple Immunogen Delivery System Based on Recombinant Bluetongue Virus-Derived Tubules

AbstractThe development of particular vector systems for the presentation of immunogenic epitopes provides a powerful approach for the delivery of antigens. These include the core-like particles formed by recombinant bluetongue virus (BTV) capsid proteins VP3 and VP7 synthesized in insect cells by recombinant baculoviruses. Previously we have reported localization of an antigenic site on the surface of tubular structures formed by the nonstructural protein NS1 of BTV, and their potential use for epitope presentation. In this study foreign sequences ranging from 44 to 116 aa in length and representing 44 aa sequence fromClostridium difficiletoxin A, 48 aa of the hepatitis B virus preS2 region, and the whole of bovine leukemia virus p15 protein were inserted at the C-terminus of BTV-10 NS1. The chimeric NS1 genes were expressed using recombinant baculoviruses and the ability of the mutated NS1 proteins to form tubules was investigated. All chimeric constructs formed tubular structures which carried the foreign antigenic sequences exposed on the surface of the tubules and were highly immunogenic. When Sf cells were coinfected with three recombinant baculoviruses expressing chimeric NS1 proteins with different epitopes, their simultaneous assembly into the same tubule was demonstrated. This observation opens up the possibility of using recombinant NS1 tubules as carriers for the delivery of multiple epitopes
DNA Sequences Responsible for Specificity of DNA Packaging and Phage Growth Interference of Bacteriophages T3 and T7

AbstractT3 and T7 phages package recombinant plasmids carrying DNA necessary for DNA packaging (thepacsequences) of T3 and T7, respectively. Packaging is specific between T3 and T7. Thepacsequence has a bipartite structure, consisting of target sequences for processing of concatemeric DNA (pacC) and its left side flanking sequence containing a promoter for phage RNA polymerase (pacB). To determine the sequences responsible for the specificity of plasmid DNA packaging, plasmids chimeric for thepacB andpacC sequences of T3 and T7 were constructed. Analysis of packaging of the chimeric plasmid DNAs showed thatpacB is responsible for the packaging specificity of T3 and T7 DNAs. Plasmids carrying the genetic right end of T3 and T7 DNA interfered with the growth of T3 and T7 phages, respectively. Interference was specific between T3 and T7.pacB and sequences betweenpacB andpacC, but notpacC, were responsible for the interference. The specificity of interference was determined bypacB and sequences responsible for interference were partially defined
AnAutographa californicaNucleopolyhedroviruslef-2 Mutant: Consequences for DNA Replication and Very Late Gene Expression

AbstractIn order to define factors involved in very lateAutographa californicanucleopolyhedrovirus (AcMNPV) gene function, random mutagenesis of a baculovirus recombinant (AcUW1.lacZ) by 5′-bromodeoxyuridine treatment was performed. Five viruses were selected with deficiencies in very late gene expression. These were characterized by complementation analysis. One mutant virus, VLD1, was found to be completely deficient in very late gene function. This virus could be complemented by a helper virus to express the very late genes, suggesting that the mutant virus was defective in an activator of very late gene expression. Further studies revealed that the replication of VLD1 was temporally delayed when compared to wild-type virus. The mutation in VLD1 was mapped to a subfragment of theEcoRI-I region of the AcMNPV genome between 0 and 5 map units. Sequence analysis revealed the presence of point mutations in ORF2 and inlef-2. Further mapping experiments demonstrated that only replacement of the point mutation inlef-2 with a wild-type sequence could restore VLD1 to a normal phenotype. Previous studies have suggested that thelef-2 gene product is involved in DNA replication. This was investigated by comparison of DNA replication in wild-type- and VLD1-infected cells. It was found that the mutation in thelef-2 gene of VLD1 did not have an effect on DNA replication. It is proposed thatlef-2 may play a dual role, both in DNA replication and very late gene expression
Selective Expression of a Subset of Measles Virus Receptor-Competent CD46 Isoforms in Human Brain

AbstractThe human cell surface protein CD46 is the main measles virus (MV) receptor. We analyzed the CD46 isoforms expressed in the brain of three patients who died with persistent MV infections and in an unaffected brain. Complete CD46 cDNAs were produced and found to code exclusively for CD46 isoforms with cytoplasmic tail 2. Selective expression of tail 2 isoforms was shown in a second control brain by Western blots with antibodies specific for each of the cytoplasmic tails. Binding of purified MV particles and virus-dependent cell fusion were tested after transient expression of brain-derived CD46 proteins in mouse cells. All the brain-derived proteins mediated MV binding and virus-dependent fusion. Isoforms containing both serine/threonine/proline (STP)-rich domains were more active in virus binding, whereas isoforms with only one STP domain were more efficient in mediating fusion
Immunodominant E2 (gp53) Sequences of Highly Virulent Bovine Viral Diarrhea Group II Viruses Indicate a Close Resemblance to a Subgroup of Border Disease Viruses

AbstractThe genes for the E2 envelope protein, which elicits virus-neutralizing antibodies, from members of the newly described group II of bovine viral diarrhea viruses (BVDVs) were cloned and sequenced. These BVDVs included a thrombocytopenic strain from the United States, a fetal bovine serum contaminant, a strain from Western Canada, and two highly virulent strains, causing high mortality rates, from Quebec. The nucleotide and amino acid sequences of these E2s had only a 60–65% homology with group I BVDV E2s but >90% homology with the E2 of a subgroup of sheep border disease viruses. The E2 gene of the NADL strain was expressed and monospecific antibodies were raised in calves and rabbits. The virus-neutralizing titers of these antisera were 15- to 80-fold lower for the heterologous group of BVDVs as compared to those for the homologous BVDVs
Oliveros Virus: A Novel Arenavirus from Argentina

AbstractDuring the past few decades several newly recognized rodent-borne arenaviruses have been shown to be associated with severe hemorrhagic fever cases in South America. Changes in ecology and farming practices throughout the region have increased the concern over the potential public health threat posed by such emerging virus diseases. Oliveros (OLV) virus is a recently discovered arenavirus of the rodentBolomys obscurusin Argentina. Genetic analysis of the small genomic RNA segment, which encodes the nucleocapsid protein and the envelope glycoproteins, shows that Oliveros is a novel, phylogenetically distinct member of theArenaviridaefamily which differs in nucleotide sequence from the previously characterized members by approximately 35% or more. Despite this level of diversity, OLV virus possesses the same ambisense genome structure and many overall RNA and protein features in common with other arenaviruses. These data represent an important first step in the development of specific immunological and PCR diagnostic reagents to allow assessment of the prevalence and disease potential of this virus