pinecone/core-2020-05-10-deduplication huggingface dataset:
Intact Eukaryotic Initiation Factor 4G Is Required for Hepatitis A Virus Internal Initiation of Translation

AbstractThe requirements for optimal activity of the hepatitis A virus (HAV) internal ribosome entry segment (IRES) differ substantially from those of other picornavirus IRESes. One such difference is that, to date, the HAV IRES is the only one whose efficiency is severely inhibited in the presence of the picornaviral 2A proteinase. Here we describe experiments designed to dissect the mechanism of proteinase-mediated inhibition of HAV translation. Using dicistronic mRNAs translatedin vitro,we show that the HAV IRES is inhibited by the foot-and-mouth disease virus Lb proteinase, as well as by the human rhinovirus 2A proteinase. Furthermore, using mutant Lb proteinase, we demonstrate that proteolytic activity is required for inhibition of HAV IRES activity. Translation inhibition correlated closely with the extent of cleavage of the one identified common cellular target for the 2A and Lb proteinases, eukaryotic initiation factor (eIF) 4G, a component of the eIF4F cap-binding protein complex. Total rescue of HAV IRES activity was possible if purified eIF4F was added to translation extracts. In contrast, if the added eIF4F contained cleaved eIF4G, no rescue of HAV IRES activity was evidenced. Thus the HAV IRES requires intact eIF4G for activity. This is unique among the picornavirus IRESes studied to date and may help explain why HAV does not inhibit host cell translation during viral infection
Comparison of the Rotavirus Gene 6 from Different Species by Sequence Analysis and Localization of Subgroup-Specific Epitopes Using Site-Directed Mutagenesis

AbstractThe nucleotide sequence of gene 6 encoding the rotavirus major capsid protein VP6 of EDIM strain (EW) was determined and compared to that of 20 previously reported strains with known subgroup specificities. Multiple alignments of amino acid sequences exhibited a high level of sequence conservation (87 to 99.2%). Site-specific mutagenesis experiments were undertaken to localize regions involved in subgroup specificity. Amino acid positions 305, 315, and a region 296–299 (or 301 for equine strain H-2) were identified as contributing to subgroup epitopes. A single amino acid mutation at position 305 or 315 was sufficient to change the subgroup specificity of EW VP6 protein from non I/II to subgroup I- or subgroup II-like, respectively. Mutation at these sites may be another important mechanism for subgroup variation, along with gene reassortment
Deletion Mutants of the Herpes Simplex Virus Type 1 UL8 Protein: Effect on DNA Synthesis and Ability to Interact with and Influence the Intracellular Localization of the UL5 and UL52 Proteins

AbstractThe herpes simplex virus type 1 (HSV-1) helicase-primase, an essential component of the viral DNA replication machinery, is a trimeric complex of the virus-coded UL5, UL8, and UL52 proteins. An assembly of the UL5 and UL52 subunits retains both enzymic activities, and the UL8 protein has been implicated in modulating these functions, facilitating efficient nuclear uptake of the complex and interacting with other viral DNA replication proteins. To further our understanding of UL8, we have constructed plasmids expressing mutant proteins, truncated at their N- or C-termini or lacking amino acids internally, under the control of the human cytomegalovirus major immediate-early promoter. Deletion of 23 amino acids from the N-terminus or 33 from the C-terminus abolished the ability of UL8 to support DNA replication in transient transfection assays. None of the UL8 mutants tested exhibited a strong dominant negative phenotype in the presence of the wild-type product, although some inhibition of replication was observed with mutants lacking 165 N-terminal or 497 C-terminal amino acids. The ability of the UL8 mutants to facilitate efficient nuclear localization of UL52 in the presence of coexpressed UL5 was examined by immunofluorescence. Selected mutants were also expressed by recombinant baculoviruses and tested for interaction with UL5 and UL52 in immunoprecipitation assays. The replicative ability of the mutants was found to correlate with their ability to localize UL52 to the nucleus, but not their interaction with UL5 and UL52. This property precluded the identification of any region of UL8 important for its presumed nuclear functions
Differential Effects on HIV-1 Gene Regulation by EBV in T Lymphocytic and Promonocytic Cells Transduced to Express Recombinant Human CR2 (CD21)

AbstractA panel of human hematopoietic cell lines was genetically engineered to express recombinant complement receptor 2 (CR2 or CD21), which is also the Epstein–Barr virus (EBV) receptor. The panel was composed of SupT1, J1.1, and U1.HIV cells. The latter is a promonocytic cell line, whereas the other two are T lymphocytic cell lines. J1.1 and U1.HIV cells are latently infected by human immunodeficiency virus type 1 (HIV-1). These three cell lines were transduced with a murine leukemia virus (MLV)-based retroviral vector system. CR2 was efficiently and consistently expressed on the cell membranes, conferring enhanced susceptibility to EBV infection. The efficient expression of recombinant CR2 in cell lines of hematopoietic origin allowed for study of the interaction between EBV infection and HIV-1 gene regulation in suitable cell-culture models. The effects of EBV and HIV-1 coinfection results were cell-type dependent. In the two T lymphocytic cell lines, HIV-1 expression was rapidly and persistently down-regulated by EBV. Conversely, in the promonocytic cell line U1.HIV-CR2, HIV-1 expression was transiently enhanced by EBV. The EBV and HIV-1 coinfection result in U1.HIV-CR2 cells is potentially important, as the activation of HIV-1 gene expression in monocyte-like cells may play a crucial role in the mechanism of CD4+T cell depletion by apoptosis. Therefore, the U1.HIV-CR2 cell line may represent a useful cell-culture system to study the synergism between EBV and HIV-1 in inducing apoptosis in primary CD4+T cells
Failure to Complement Infectivity of EBV and HSV-1 Glycoprotein B (gB) Deletion Mutants with gBs from Different Human Herpesvirus Subfamilies

AbstractGlycoprotein B (gB) is conserved among the herpesvirus family which infects a broad range of species. To investigate the functional homology of human α-herpesviruses, β-herpesviruses, and γ-herpesviruses gB proteins, complementation studies were performed with gB genes from each subfamily member using EBV gp110 (EBV gB homologue) and HSV-1 gB null mutants. Neither the α-herpesvirus HSV-1 gB gene nor the β-herpesvirus HCMV gB gene were able to complement the gp110 null mutant. Conversely, neither the β-herpesvirus HCMV gB or the γ-herpesvirus EBV gp110 gene were able to complement HSV-1 gB null mutants. To further investigate functional domains of EBV gp110 and HSV-1 gB, gB-gp110 chimeric proteins were constructed. Surprisingly, none of the chimeric proteins were able to complement either HSV-1 gB null mutants or EBV gp110 null mutants. These results demonstrate that there is not sufficient functional homology between the different gBs to allow complementation in other subfamily members of the herpesvirus family
Intracellular Processing of Pseudorabies Virus Glycoprotein M (gM): gM of Strain Bartha Lacks N-Glycosylation

AbstractGenes encoding homologs of the herpes simplex virus type 1 UL10 product, glycoprotein M, are conserved in all herpesviruses investigated so far. Recently, we identified pseudorabies virus (PrV) gM as a 45-kDa structural component of purified virions. A gM−PrV mutant could be propagated in cell culture, albeit at lower titers and with delayed penetration kinetics. Thus, gM has a nonessential but modulatory function in PrV infection. PrV gM is modified by addition of an N-linked glycan at a consensus sequence located between the predicted first and second hydrophobic region of the protein. This N-glycosylation site is conserved in all gM homologs sequenced so far, indicating an important functional role. To analyze intracellular processing of PrV gM, Western blot analyses were performed. In PrV-infected cells, mature 45-kDa gM as well as 33- and 35-kDa precursor forms were detectable. Presumably dimeric 90- and 70-kDa proteins were also observed. The 33- and 35-kDa proteins represent nonglycosylated and glycosylated precursors as shown by endoglycosidase digestions. Investigation of several PrV strains revealed that the UL10 product of PrV strain Bartha, an attenuated virus used as vaccine, was not modified by N-glycosylation. Sequence analysis showed that the N-glycosylation consensus sequence was altered from NDT to NDA, which resulted in loss of the N-glycosylation signal. To our knowledge, this is the only gM homolog identified so far which is not N-glycosylated. To investigate whether this form of the protein is functionally competent, the UL10 gene of strain Bartha was inserted into PrV strain Kaplan by substitution of the wild-type UL10 gene. The resulting recombinant expressed a UL10 protein lacking N-glycans.In vitroreplication analyses did not reveal any difference in virus production, but plaque size and penetration kinetics were slightly reduced. In summary, we show that wild-type gM is modified by N-glycosylation at one conserved site. However, although this site is highly conserved throughout the herpesviruses, loss of N-glycans due to mutation of the consensus sequence had only a minor effect on propagation of PrV in cell culture
Characterization of Events during the Late Stages of HPV16 Infectionin VivoUsing High-Affinity Synthetic Fabs to E4

AbstractHPV late gene expression is initiated as an infected basal cell migrates through the differentiating layers of the epidermis, resulting in the onset of vegetative viral DNA replication and the expression of viral late proteins. We have used a large synthetic immunoglobulin library displayed on phage (diversity 6.5 × 1010phage) to isolate three Fabs (TVG405, 406, and 407) which recognize distinct epitopes on the E4 late protein of HPV16. A C-terminal monoclonal (TVG404) was generated by hybridoma technology, and N-terminal polyclonal antiserum was prepared by peptide immunization (α N-term). The most potent antibody (TVG405) had an affinity for E4 of approximately 1.0 nM. All antibodies recognized the protein in paraffin-embedded archival material, allowing us to map events in the late stages of virus infection. Expression of E4in vivodoes not coincide with synthesis of the major virus coat protein L1, but precedes it by 1 or 2 cell layers in premalignant lesions caused by HPV16 and by up to 20 cell layers in HPV63-induced warts. In higher grade lesions associated with HPV16, E4 is produced in the absence of L1. By contrast, vegetative viral DNA replication and E4 expression correlate exactly and in some lesions begin as the infected epithelial cell leaves the basal layer. Differentiation markers such as filaggrin, loricrin, and certain keratins are not detectable in E4-positive cells, and nuclear degeneration is delayed. HPV16 E4 has a filamentous distribution in the lower epithelial layers, but associates with solitary perinuclear structures in more differentiated cells. Antibodies to the N-terminus of the protein stained these structures poorly. Our findings are compatible with a role for the HPV16 E4 protein in vegetative DNA replication or in modifying the phenotype of the infected cell to favor virus synthesis or virus release. The Fabs will be of value in the evaluation of model systems for mimicking HPV infectionin vitro
The Site-Specific Integration System of the TemperateStreptococcus thermophilusBacteriophage φSfi21

AbstractThe temperate bacteriophage φSfi21 integrates its DNA into the chromosome ofStreptococcus thermophilusstrains via site-specific recombination. Nucleotide sequencing of the attachment sites identified a 40-bp identity region which surprisingly overlaps both the 18-terminal bp of the phage integrase gene and the 11-terminal bp of a host tRNAArggene. A 2.4-kb phage DNA segment, coveringattP,the phage integrase, and a likely immunity gene contained all the genetic information for faithful integration of a nonreplicative plasmid into theattBsite. A deletion within theintgene led to the loss of integration proficiency. A number of spontaneous deletions were observed in plasmids containing the 2.4-kb phage DNA segment. The deletion sites were localized to the tRNA side of the identity region and to phage or vector DNA with 3- to 6-bp-long repeats from the border region. A similar type of deletion was previously observed in a spontaneous phage mutant
The Locus of Epstein–Barr Virus Terminal Repeat Processing Is Bound with Enhanced Affinity by Sp1 and Sp3

AbstractEBV DNA contains G-rich, repeat regions that are involved in rearrangement and recombination events including terminal repeat (TR) processing and the EBNA-2 deletion in the EBV strain P3HR-1. Cellular proteins, called terminal or tandem repeat binding proteins (TRBPs), recognize sequences at the junctions of these recombination events. In this study, using antibody supershift assays and expression of recombinant proteins, we show that Sp1 and Sp3 are the sequence-specific components of TRBP and that Ku is the nonspecific binding component. Sp1 binds other recombinogenic regions of EBV DNA, but Sp3 does not bind to the large internal repeat. The sequence GGGGTGGGG, a low affinity site for Sp1 and Sp3, is the minimal binding site within terminal repeat binding site 1 (TRBS1). However, 3′ flanking sequences in the sequence GGGGTGGGGCATGGGG augment binding of Sp1 and Sp3 so that their affinity of binding is increased approximately twofold relative to a classical high-affinity Sp1 site. EBV lytic cycle induction does not alter the abundance or binding activity of any of the three identified components of TRBP. Sp1 and Sp3 may actin transto promote EBV terminal repeat processing and possibly other viral and cellular recombination events
The 5′ Region of the Human Papillomavirus Type 31 Upstream Regulatory Region Acts as an Enhancer Which Augments Viral Early Expression Through the Action of YY1

AbstractCis-elements which control human papillomavirus early gene expression have previously been localized to sequences in the upstream regulatory region (URR) which are proximal to the E6 open reading frame. These elements include an enhancer element which functions preferentially in keratinocytes as well as promoter elements. The function of the remaining approximate 500-bp region of the URR in regulating viral expression in the high risk papillomaviruses has been largely uncharacterized. In HPV 6, a negative regulator of early expression, or silencer, has been identified in this 5′ region of the URR. In this study, we have investigated the role of the 5′ portion of the HPV 31 URR in regulating viral expression. Sequences in this region were found to exert a minimal negative effect on homologous or heterologous promoters. In contrast, a 128-bp sequence within this region was found to exhibit enhancer activity on heterologous and homologous promoters. This enhancer also augmented the activity of the previously identified minimum keratinocyte enhancer. The cellular factors, YY1 and TEF-1, were determined by DNase I footprint analysis and electrophoretic mobility shift assays (EMSAs) to bind the 128-bp enhancer. The binding of YY1 to two of these sites was found to be important for enhancer activity
Screening Human Tumor Samples with a Broad-Spectrum Polymerase Chain Reaction Method for the Detection of Polyomaviruses

AbstractPolyomaviruses induce tumors of different histological types when inoculated into experimental animals. An etiological role for this virus group in the development of malignant tumors in humans remains questionable, despite several reports demonstrating the presence of SV40, JCV, and BKV DNA in human cancers. Only two human polyomavirus types are known to date: JCV, causing progressive multifocal leukoencephalopathy (PML) under severe immunosuppression, and BKV, first isolated from the urine of a renal transplant recipient and associated with hemorrhagic cystitis. We developed a degenerate polymerase chain reaction assay in an attempt to identify additional, presently unknown human polyomavirus types that may be involved in the malignant transformation of human tissues. A large part of the gene coding for the viral capsid protein VP1 is highly conserved in nine polyomavirus types (and their strains) and was therefore selected as most suitable for the primer design. Degenerate oligonucleotide primers were deduced from four different conserved amino acid motifs in this region. Three different sets of primers were included in each test to obtain the highest sensitivity in combination with primers with the lowest degeneracy numbers. The sensitivity obtained ranged from 1 copy/cell for bovine polyomavirus to 100 copies/cell for LPV after ethidium bromide staining and was increased at least 10-fold after hybridization with a radiolabeled probe. A subsequent seminested amplification allowed for the detection of 1 copy/cell for LPV. These degenerate primers were applied to analyze bladder carcinomas, Hodgkin lymphomas, meningiomas, Kaposi tumors, and Kaposi-derived cell lines. No polyomavirus DNA sequences could be detected
Isolation of RNA Aptamers Specific to the NS3 Protein of Hepatitis C Virus from a Pool of Completely Random RNA

AbstractHepatitis C virus (HCV) is a single-stranded RNA virus and its genome is translated into a single large polyprotein. The viral-encoded NS3 protein possesses protease, nucleoside triphosphatase, and helicase activities. Since these activities appear to be important for viral replication, efforts are being made to identify compounds that might inhibit the enzymatic activities of NS3 and serve as potential anti-HCV agents. We used a genetic selection strategyin vitroto isolate, from a pool of completely random RNA (120 random bases), those RNA aptamers that could bind to NS3. After six cycles of selection and amplification, 14% of the pooled RNAs could bind specifically to the NS3 protein. When the aptamers in the pool (cycle 6) were analyzed for binding and inhibition of the proteolytic activity of NS3 with the NS5A/NS5B peptide as substrate (S1), two aptamers, designated G6–16 and G6–19 RNA, were found to inhibit NS3in vitro.Kinetic studies of the inhibition revealed that the aptamer G6–16 inhibited the NS3 protease with an inhibitory constant (Ki) of 3 μM.We also analyzed aptamers G6–16 and G6–19 for their action with a longer protein substrate (amino acid region 2203–2506) and found that these aptamers efficiently inhibited the proteolytic activity of NS3. In addition, both G6–16 and G6–19 aptamers were found to inhibit the helicase activity of NS3. Since these aptamers possesses dual inhibitory function for NS3, they could prove to be useful as anti-HCV drug leads
Definition of the Primary Structure of Hepatitis B Virus (HBV) pre-S Hepatocyte Binding Domain Using Random Peptide Libraries

AbstractThe pre-S-specific monoclonal antibody MA 18/7 has been shown to inhibit the binding of HBV to HepG2 cells and liver membranes. This antibody can thus be used to identify the critical residues of the pre-S region involved in the hepatocyte-binding domain. Using overlapping 7-mer peptides representing the pre-S region of HBV, the epitope recognized by MA 18/7 was shown to contain sequences from both the pre-S1 and pre-S2 regions, thus indicating that the hepatocyte-binding domain is conformationally dependent. To further characterize the primary structure of the hepatocyte-binding domain on the pre-S protein, a phage-displayed 15-mer peptide library and a 8-mer solid phase peptide library were used to analyze the fine specificity of the monoclonal antibody MA 18/7. Several mimotopes were identified with the phage-displayed peptide library, the majority of which possess a central motif with at least three identical residues present within the native pre-S1 sequence. No significant consensus sequences were found when these mimotopes were compared to the pre-S2 sequence. Mimotopes identified using the solid-phase peptide library also contained a similar motif. All phage mimotopes and a single mimotope from the solid-phase peptide library competed with recombinant HBsAg particles containing the pre-S1 region for binding to MA 18/7. Mouse antisera raised against four mimotopes from the phage display library reacted with HBsAg particles containing pre-S sequences. The data show that the structure of the pre-S molecule around the conserved DPAF motif in the pre-S region may have a functional role in binding HBV to cellular receptors, and that the central motif identified in mimotopes of this region may offer a novel strategy target for the improvement of existing hepatitis B vaccines which, at present, are mostly devoid of pre-S specificities
Induction of Intracellular Membrane Rearrangements by HAV Proteins 2C and 2BC

AbstractHepatitis A virus (HAV) is distinguished from other picornaviruses by its slow and relatively poor, noncytopathic growth in cultures of mammalian cells. The 2C and 2BC proteins of HAV have been implicated in the determination of virus growth in cultured cells. The homologous proteins from other picornaviruses, such as poliovirus, have been demonstrated to exhibit multiple activities, such as RNA binding, nucleotide binding and NTPase, and membrane binding and reorganization. At least some of these activities are required for viral RNA replication. We report here that HAV 2C and 2BC proteins, like their poliovirus counterparts, can induce rearrangement of intracellular membranes and directly or indirectly interact with membranes. Therefore, the inefficient replication properties of HAV are not consequences of the inherent ability of 2C (2BC) to interact with membranes. The effect of 2C (2BC) protein sequences derived from a cell culture-adapted (cc) strain of HAV was compared with that of corresponding protein sequences from either a wild-type (wt) strain of HAV or a faster replicating cytopathic (cp) strain. The analysis demonstrated that mutations acquired in wt virus during adaptation to cell culture do not change dramatically either the ability of these proteins to associate with membranes and induce membrane alterations or the specific architecture of the induced membrane structures. On the other hand, 2C, but not 2BC, protein from the cp strain of HAV induced different membrane structures
Effects of Domain-Switching and Site-Directed Mutagenesis on the Properties and Functions of the VP7 Proteins of Two Orbiviruses

AbstractBased on the crystal structure of the VP7 major core protein of bluetongue virus serotype 10 (BTV-10) and that of the top domain of the VP7 protein of African horsesickness virus serotype 4 (AHSV-4), chimeras and site-directed mutants of the proteins were constructed and the products analyzed with respect to their properties and functions. Chimeras with the central upper domain of BTV-10 VP7 replaced by that of AHSV-4 VP7 (construct BAB) formed trimers, as did the converse construct (ABA). Further, both proteins exhibited the expected conformational epitopes of the constituent sequences. Using BAB VP7 it was demonstrated that residues of the upper domain of AHSV-4 VP7 contribute to the observed insolubility of the protein. By contrast, ABA VP7 protein was as soluble as wild-type BTV-10 VP7. Replacement of selected amino acid residues in the top domain (e.g., A167 by R; F209 by T) improved the solubility of BAB VP7. Since the trimeric BAB and ABA VP7 proteins did not form core-like particles (CLPs) when coexpressed with BTV VP3, it was concluded that trimerization of chimeric VP7 is not sufficient for CLP formation. When the N-terminal region of the ABA protein (aa 1–120) was replaced by the respective sequences of BTV VP7 (construct BBA), the protein aggregated and did not form CLPs with coexpressed BTV VP3, most likely due to disruption of the required contacts between the N- and C-terminal regions of the bottom domain, leading to incorrect folding of the chimera