pinecone/core-2020-05-10-deduplication huggingface dataset:
Characterization of P91, a Protein Associated with Virions of anOrgyia pseudotsugataBaculovirus

AbstractPolyclonal antiserum produced against preoccluded virions from theOrgyia pseudotsugatamultinucleocapsid nuclear polyhedrosis virus (OpMNPV) was used to screen an OpMNPV λgt11 expression library. The insert from one of the immunoreactive phage isolates hybridized to OpMNPVorf86(p91), a 2460-bp (819 amino acids) open reading frame that encodes a predicted protein of 91 kDa. Antibodies generated against a maltose binding protein–P91 fusion detected a band of approximately 91 kDa on Western blots of extracts of OpMNPV-infectedLymantria disparcells. This band was first observed at 18 hr p.i. and was present at all later time points. Similar results using this antiserum were seen with a time course ofAutographa californica-infectedSpodoptera frugiperdacells. Localization of P91 by confocal immunofluorescence microscopy showed that the protein was concentrated near the nuclear membrane and at late times p.i. was most concentrated near polyhedra. Immunoelectron microscopy indicated that P91 was present in both the capsid and envelope surrounding the capsid of occlusion-derived virions. Fractionation studies employing NP-40 and Western blot analysis indicated that P91 was associated with the capsid structure
Genetic Analysis of Internal Ribosomal Entry Site on Hepatitis C Virus RNA: Implication for Involvement of the Highly Ordered Structure and Cell Type-Specific Transacting Factors

AbstractHepatitis C virus (HCV) carries an internal ribosomal entry site (IRES) within the 5′ portion of the RNA. To identify structures that influence efficiency of the translation initiation, relative activities of modified IRESs were examined by using engineered bicistronic mRNAs, between the two cistrons of which various mutant IRESs were inserted. An IRES derived from genotype 2b is at least two times more efficient than one from genotype 1b in cultured cells. Activity ratios of genotype 2b IRES to 1b IRES differ in magnification among cultured cells, suggesting the difference in assortment of IRES-related host factors among individual cell types. Recombinant IRESs between the genotypes show similar or higher activities compared with 2b IRES in cell-free systems and show intermediate activities in cultured cells. Patterns of relative activities of those IRESs indicate that the IRES activity is not regulated by defined structure(s), although a cluster of different nucleotides is observed in the genome region of nucleotides 176–224 between the two alleles. The results suggest that a highly ordered structure formed by the entire 5′ portion of the RNA is important for the IRES activity. The 5′ border of HCV IRES was examined by using a series of deletion RNAs in various systems. The results strongly suggest that the border resides between nucleotide positions 28 and 45. Patterns of relative activities of the deletion IRESs differ in translation systems or cell types. These results imply that interactions of HCV RNA with the related transacting factor(s) may differ in the translation systems or cell types
Cell Attachment and Mouse Virulence of Echovirus 9 Correlate with an RGD Motif in the Capsid Protein VP1

AbstractThe recently analyzed sequences of the nonpathogenic prototype strain Hill and the mouse-virulent strain Barty of the human echovirus 9 differ particularly in an insertion coding for an RGD motif at the C-terminus of the capsid protein VP1 in the genome of strain Barty. To investigate molecular determinants of virulence, we generated a panel of recombinant viruses derived from cDNA clones of strains Hill and Barty. In this communication, we show that the mouse-pathogenic character of strain Barty correlates with a 310-aa segment including the RGD motif. By mutating the RGD to an RGE tripetide, the infectivity of the resulting echovirus 9 clones for GMK cells is lost. Furthermore, we could show that synthetic peptides containing the RGD sequence influence binding of mouse-virulent echovirus 9 strains to GMK cells, whereas binding of apathogenic strains is not affected. These results suggest that the RGD motif is a significant factor affecting pathogenicity of echovirus 9 strains
U5 Region of the Human Immunodeficiency Virus Type 1 Long Terminal Repeat Contains TRE-like cAMP-Responsive Elements That Bind both AP-1 and CREB/ATF Proteins

AbstractActivating protein-1 (AP-1) binding phorbol ester responsive elements (TRE) are located downstream of the transcription initiation site in the U5 region of the human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR). These downstream sequence elements, termed DSE, can bind cFos and JunD and transmit protein kinase C (PKC) activation signals to the LTR. Further studies suggested the DSE might also bind AP-1-related proteins of the CREB/ATF family. Since enhanced HIV-1 expression is associated with activation of the cAMP-dependent protein kinase A (PKA) signaling pathway, we determined whether binding of CREB/ATF proteins to the DSE mediate cAMP/PKA activation of the HIV-1 LTR. In the present study, DSE binding complexes in nuclear protein extracts from colonic epithelial cells are shown to contain ATF-1, ATF-2, and CREB and transfection of either an ATF-2 or PKA expressing plasmid transactivated the DSE. Cholera toxin (Ctx), a potent activator of the cAMP/PKA pathway, increased HIV-1 virus production from a latently infected promonocytic cell line, U1. Ctx increased LTR promoter activity and increased the CREB content of DSE binding complexes. Transfection of U1 cells with a series of mutant LTR reporter constructs demonstrated that the Ctx response was in large part mediated by the DSE. The Ctx response was also mediated by a heterologous promoter containing multiple TRE sites. Nuclear protein extracts from a T-cell line infected by HIV-1 contained higher levels of CREB/ATF proteins and manifested increased CREB/ATF binding activity. Collectively, these results indicate the DSE are TRE-like cAMP responsive elements that bind both AP-1 and CREB/ATF permitting induction of the HIV-1 LTR by both PKC and PKA activation signals
Characterization of the Lysogeny DNA Module from the TemperateStreptococcus thermophilusBacteriophage φSfi21

AbstractPhage φSfi21, the only temperateStreptococcus thermophilusphage from our phage collection, showed extensive DNA homology with virulent phages from lytic group I. Southern blot hybridizations demonstrated that the φSfi21-specific DNA was clustered in an approximately 6.6-kb-long region, the putative lysogeny module. Sequence analysis and database research identified an integrase within this module; orf 203 with homology to an anonymous orf 258 from the temperate lactococcal phage BK5-T; orf 127 and orf 122 with weak homology to the N- and C-terminal parts, respectively, of the cI-like repressor from lactococcal phages Tuc2009 and BK5-T; orf 75 with homology to a repressor protein from lambdoid phage 434 and an anti-repressorantwith homology to phage P1. The molecular arrangement of the predicted orfs in phage φSfi21 was very similar to that of the lactococcal phage BK5-T. The transition from φSfi21-specific DNA into DNA shared with virulent phages was abrupt and flanked at one side by notable DNA repeats. Sequence analysis identified a holin protein to the left of the lysogeny module. A site-specific deletion of 2.4 kb, which reproducibly transformed φSfi21 into a lytic phage, was localized in the lysogeny module. It was flanked at both sides by conspicuous DNA repeats. One repeat region reflected the DNA around theattPsite, while the other reflected the putative genetic switch region between repressor and anti-repressor genes.S. thermophilushost Sfi1 transformed with a plasmid containingintand orf 203 showed resistance to superinfection by heterologous phages, but not by the homologous φSfi21. Part of theintgene could be deleted without loss of this activity, while a deletion in orf 203 resulted in loss of the phage resistance. We speculate on the possibility of a bipartite immunity system for the control of lysogeny in φSfi21
In VivoLongitudinal Analysis of a Dominant TCR Repertoire Selected in Human Response to Influenza Virus

AbstractRecent studies have demonstrated biased usage of TCR Vβ17 and a high degree of diversity in Jβ usage within the influenza virus matrix epitope (M.58-66)-specific CTL response. In contrast, in the course of a study on the cellular response to influenza A virus, we found preferential usage of Vβ17-Jβ2.2 rearrangement in an individual with an unexpectedly high number of CTL precursors (CTLp). We took advantage of such situation to study the longitudinal repertoire of the CD8+T cell precursors. By limiting dilution analysis combined with the use of a clonotypic primer corresponding to the CDR3 region of this matrix-specific TCR Vβ chain, the influenza-specific CTLp were shown to be stable for a period of 5 years. Overall, our results show that virus-specific CTLp can be directly monitoredin vivoby molecular fingerprinting withoutin vitrorestimulation. These findings might be extremely important for evaluation of the specific immune response to a given human pathogen
Mutations in CR1 of E1A 12S Yield Dominant Negative Suppressors of Immortalization and the Lytic Cycle

AbstractThe Adenovirus 5 E1A 12S gene is responsible for the establishment of immortalization of primary cells by Adenovirus. We have identified two mutants of 12S (30K and NTdl814), which, when coexpressed with wild-type 12S in primary baby rat kidney cells, were capable of suppressing the immortalizing function of the wild-type 12S gene, even when the mutant proteins were expressed at levels lower than wild type. 30K and NTdl814 did not affect the ability of the coexpressed 12S to activate the cell cycle, but have a suppressive effect on 12S-induced DNA synthesis and proliferation at late times in the immortalization pathway. Both the dominant negative mutants have a deletion in conserved region (CR)1 in the first exon of E1A, which encompasses one of the pRb-family binding regions. However, the mutants did not affect the binding of cellular proteins to full-length 12S. A suppressive effect on wild-type 12S was not observed with mutants that have lost any other region or function. In addition, expression of 30K, which is equivalent to the protein encoded by the 10S mRNA of E1A, inhibited E1A function in lytic cycle. Thus, loss of the CR1 seems to be a prerequisite for a mutant to have a dominant negative effect on E1A functions
Indicator Cell Lines for Detection of Primary Strains of Human and Simian Immunodeficiency Viruses

AbstractCCR5 and CXCR4 are the two major coreceptors that have been identified for human immunodeficiency virus (HIV) entry. We have modified several β-galactosidase-based HIV indicator cell lines to express CCR5 and/or CXCR4. Using these new reagents, we have been able to detect all primary isolates tested using one or both of these cell lines. However, there is large variation in the absolute viral infectivity among primary strains. Furthermore, all HIV strains are capable of causing syncytia in the indicator cells when the coreceptor is present regardless of whether they had previously been characterized as “syncytia-inducing” or “non-syncytium-inducing.
The Genome of Molluscum Contagiosum Virus: Analysis and Comparison with Other Poxviruses

AbstractAnalysis of the molluscum contagiosum virus (MCV) genome revealed that it encodes approximately 182 proteins, 105 of which have direct counterparts in orthopoxviruses (OPV). The corresponding OPV proteins comprise those known to be essential for replication as well as many that are still uncharacterized, including 2 of less than 60 amino acids that had not been previously noted. The OPV proteins most highly conserved in MCV are involved in transcription; the least conserved include membrane glycoproteins. Twenty of the MCV proteins with OPV counterparts also have cellular homologs and additional MCV proteins have conserved functional motifs. Of the 77 predicted MCV proteins without OPV counterparts, 10 have similarity to other MCV proteins and/or distant similarity to proteins of other poxviruses and 16 have cellular homologs including some predicted to antagonize host defenses. Clustering poxvirus proteins by sequence similarity revealed 3 unique MCV gene families and 8 families that are conserved in MCV and OPV. Two unique families contain putative membrane receptors; the third includes 2 proteins, each containing 2 DED apoptosis signal transduction domains. Additional families with conserved patterns of cysteines and putative redox active centers were identified. Promoters, transcription termination signals, and DNA concatemer resolution sequences are highly conserved in MCV and OPV. Phylogenetic analysis suggested that MCV, OPV, and leporipoxviruses radiated from a common poxvirus ancestor after the divergence of avipoxviruses. Despite the acquisition of unique genes for host interactions and changes in GC content, the physical order and regulation of essential ancestral poxvirus genes have been largely conserved in MCV and OPV
A Conserved Internal Hydrophobic Domain Mediates the Stable Membrane Integration of the Dengue Virus Capsid Protein

AbstractThe mature flavivirus capsid protein (virion C) is commonly thought to be free in the cytoplasm of infected cells and to form a nucleocapsid-like complex with genomic RNA in mature virus particles. There is little sequence conservation among flavivirus virion C proteins, but they are similar in size (e.g., 99 amino acids [aa] for the dengue-4 [DEN4] C) and in bearing a net positive charge. In addition, we noted that C contained a conserved internal hydrophobic segment (spanning aa 45–65 in the DEN4 C). Results ofin vivoexpression andin vitrotranslation of wt and mutant forms of the DEN4 virion C demonstrated that the conserved internal hydrophobic segment in the DEN C functioned as a membrane anchor domain. Signal peptide function of this segment was also suggested by its requirement for the entry of C into membranes. Virion C was integrated in membranes in a “hairpin” conformation; positively charged segments amino- and carboxy-terminal to the hydrophobic signal-anchor segment were accessible to protease digestion in the “cytoplasm.” The net positive charge in the amino-terminal extramembraneous portion of C (aa 1–44) was one determinant of the hairpin membrane orientation; a conserved positively charged residue within the hydrophobic segment (Arg-54 in the DEN4 C) was not
Entry of Mouse Hepatitis Virus into Cells by Endosomal and Nonendosomal Pathways

AbstractOBLV60 is an acid-dependent syncytium-forming variant isolated from OBL21 cells persistently infected with the pH-independent mouse hepatitis virus (MHV)-4 strain. The fusion activity of OBLV60 can be strictly regulated by controlling pH and thus provides the means to definitively examine the entry of MHV into cells by endosomal and nonendosomal pathways. Shortly after high multiplicity infection, both MHV-4 and OBLV60 were detected by electron microscopy in endosomal vesicles and were recovered from lysates of cells treated with proteinase K to remove extracellular virus. For OBLV60, but not MHV-4, exposure to lysosomotropic compounds early in infection prevented viral penetration and significantly reduced viral yields. These results suggested that both MHV-4 and OBLV60 utilized the endosomal route of entry into cells, but that MHV-4 did not require acidification of endosomal vesicles. Studies on the entry of virus through fusion at the cell surface were performed by briefly exposing surface-bound OBLV60 to a fusion-permissive pH under conditions that prevent endocytic entry. Acid treatment of surface-bound OBLV60 caused a significant increase in the yields of virus produced in cultures of fusion-sensitive Sac− or DBT cells, demonstrating entry of virus by fusion at the cell surface. No measurable increase in virus production was detected with acid treatment of OBLV60 bound to OBL21 cells, suggesting that entry at the cell surface does not occur in these cells, which are resistant to MHV-induced syncytia formation. These results raise interesting questions concerning how mechanisms of MHV entry influence the selection of fusion variants
SV40 and Adenovirus May Act as Cocarcinogens by Downregulating GlutathioneS-Transferase Expression

AbstractWe have discovered a novel function of the SV40 T antigen and the adenovirus E1A proteins: the ability to downregulate the endogenous expression of an important detoxification enzyme, glutathioneS-transferase alpha (GSTα). GSTα mRNA is much less abundant in rat and human cells that express SV40 T antigen than in the parental cell lines. This GSTα downregulation does not require expression of SV40 small t antigen or complex formation between large T antigen and p53, p300, or the pRb family of proteins. As might be predicted, cells that express SV40 T antigen are more sensitive than normal cells to alkylating drugs, which GSTα is known to detoxify. Finally, GSTα expression is also downregulated in cells that express the adenovirus E1A proteins. We propose that by downregulating GSTα expression and inactivating p53 function, SV40 and adenovirus may contribute to the initiation of, or the progression toward, malignancy. Thus, in their quest to establish persistent infections, these viruses may inadvertently make the cellular environment more permissive for tumorigenesis
Analysis of Six DNA Components of the Faba Bean Necrotic Yellows Virus Genome and Their Structural Affinity to Related Plant Virus Genomes

AbstractFaba bean necrotic yellows virus (FBNYV) has a multicomponent circular ssDNA genome. In addition to a previously described genome component (C1) coding for a replicase-associated protein (Rep), five further components (C2 to C6) have now been identified. Each of the six components is about 1 kb in size, contains one major open reading frame (ORF) in the virion sense with a TATA box and polyadenylation signal, and has a noncoding region containing a highly conserved sequence possibly forming a stem–loop structure. Similar to C1, C2 encodes another putative Rep of 33.1 kDa, which is closely related to the Rep of banana bunchy top virus (BBTV). Based on bacterial expression and immunoblot analysis, the ORF of C5 encodes the capsid protein (CP) with a deduced molecular mass of 19 kDa. The FBNYV CP shares the highest amino acid (aa) identity (56.2%) with that of subterranean clover stunt virus (SCSV). The ORF of C4 potentially codes for a hydrophobic protein which appears to be structurally and functionally similar to the BBTV-C4 and SCSV-C1 proteins. No protein sequence similarities were found in databases for the C3 and C6 ORFs of FBNYV. FBNYV is clearly distinct from any known virus but is taxonomically related to BBTV and SCSV
A Role for Baculovirus GP41 in Budded Virus Production

AbstractInsect cells infected withtsB1074, a temperature-sensitive mutant ofAutographa californicanuclear polyhedrosis virus, exhibit a “single-cell-infection” phenotype whereby the infection progresses through the very late phase culminating in occlusion body formation, but neighboring cells do not become infected. Marker rescue mapping and DNA sequencing correlated a single nucleotide substitution within the baculovirusgp41gene with the temperature-sensitive phenotype oftsB1074. The product of thegp41gene, GP41, is an O-glycosylated protein found in occluded but not budded virions [M. Whitford and P. Faulkner (1992)J. Virol.66, 3324–3329]. However, budded virus was not produced intsB1074-infected cells at the nonpermissive temperature of 33°, indicating an additional role for GP41 in budded virus formation. Electron microscopy revealed that nucleocapsids were produced but retained in the nucleus oftsB1074-infected cells at 33°. Thus, GP41 was required for the egress of nucleocapsids from the nucleus in the pathway of budded virus synthesis
Kinetics of Cytokine mRNA Expression in the Central Nervous System Following Lethal and Nonlethal Coronavirus-Induced Acute Encephalomyelitis

AbstractThe potential role(s) of cytokines in the reduction of infectious virus and persistent viral infection in the central nervous system was examined by determining the kinetics of cytokine mRNA expression following infection with the neurotropic JHM strain of mouse hepatitis virus. Mice were infected with an antibody escape variant which produces a nonlethal encephalomyelitis and compared to a clonal virus population which produces a fulminant fatal encephalomyelitis. Infection with both viruses induced the accumulation of mRNAs associated with Th1- and Th2-type cytokines, including IFN-γ, IL-4, and IL-10. Peak mRNA accumulations were coincident with the clearance of virus and there was no obvious differences between lethally and nonlethally infected mice. TNF-α mRNA was induced more rapidly in lethally infected mice compared to mice undergoing a nonfatal encephalomyelitis. Rapid transient increases in the mRNAs encoding IL-12, iNOS, IL-1α, IL-1β, and IL-6 occurred following infection. Nonlethal infections were associated with increased IL-12, IL-1β, and earlier expression of IL-6, while lethal infections were associated with increased iNOS and IL-1α mRNA. These data suggest a rapid but differential response within the central nervous system cells to infection by different JHMV variants. However, neither the accumulation nor kinetics of induction provide evidence to distinguish lethal infections from nonlethal infections leading to a persistent infection. Accumulation of both Th1 and Th2 cytokines in the central nervous system of JHMV-infected mice is consistent with the participation of both cytokines and cell immune effectors during resolution of acute viral-induced encephalomyelitis
Characterization of Human Endogenous Retrovirus Type K Virus-like Particles Generated from Recombinant Baculoviruses

AbstractThe family of human endogenous retrovirus type K (HERV-K) comprises members with long open reading frames (ORF) for retroviral proteins. The existence of a biologically active provirus with replicative capacities has not yet been demonstrated. To confirm the assumption that HERV-K codes for the previously observed retrovirus-like particles (human teratocarcinoma-derived virus, HTDV) in human teratocarcinoma cells, we have constructed recombinant full-length HERV-K cDNA-based baculoviruses withgag, pro, pol,andenvORFs. Two viral constructs were used for infections of insect cells, one bearing 67 bp of the 5′ untranslated region upstream of the 5′ splice donor (SD) site and of the retroviral genes, the second omitting the SD sequence. For both recombinant viruses, indirect immunofluorescence and laser scan analyses revealed expression of HERV-K Gag protein. Electron microscopy studies demonstrated efficient production of virus-like particles (VLPs) at the cytoplasmic cell membranes. These VLPs are morphologically identical with the HTDV phenotype. In immunoelectron microscopy of ultrathin frozen sections, anti-HERV-K Gag antibodies specifically reacted with HERV-K VLPs. In Western blots, in addition to the 76-kDa precursor protein, the putative major core protein with an apparent molecular mass of 32 kDa exhibited predominant immunoreactivity with anti-Gag antiserum. In contrast, neither HERV-K Env nor cORF proteins could be detected due to inefficient mRNA splicing. Purified particles from insect cell culture supernatants tested in an ultrasensitive reverse transcriptase assay revealed weak polymerase activity. The data demonstrate that HERV-K codes for retroviral particles of the HTDV phenotype