pinecone/core-2020-05-10-deduplication huggingface dataset:
DNA Sequence Requirements for the Activation of a CATAAA Polyadenylation Signal within the Hepatitis B Virus X Reading Frame: Rapid Detection of Truncated Transcripts

AbstractTruncated hepatitis B virus transcripts terminating downstream of a cryptic CAUAAA polyadenylation signal within the HBx open reading frame have previously been identified in tissue samples from two patients with hepatocellular carcinoma (Hilgeret al.,1991,J. Virol.65, 4284–4291). In this study an HBx expression plasmid was systematically deleted in order to elucidate the DNA sequence context which is required for the conversion of the usually inactive CAUAAA motif into a functional polyadenylation signal. Deletions were made progressively on a stretch of viral DNA which, seen on the transcript level, started downstream of the established UAUAAA polyadenylation signal and proceeded to the cryptic CAUAAA motif. The plasmid constructs obtained were used to transfect cells of the HepG2 line. The analysis of newly synthesized RNA via an RNase protection assay revealed termination downstream of the CAUAAA motif following the removal of GU-rich auxiliary sequences downstream of the poly(A) addition site of the UAUAAA signal. Similar results were obtained when an anchored oligo(dT) primer which recognizes selectively truncated RNA was used for the differential, RT/PCR-mediated amplification of 3′-ends. Thus it could be documented in two ways that inactivation or removal of the UAUAAA signal rendered the CAUAAA motif functional as a poly(A) signal. On the basis of the results obtained, we suggest that chromosomally integrated viral DNA on which the TATAAA motif is removed may constitute a template for truncated as well as for virus/cell hybrid transcripts. We also suggest the use of anchored oligo(dT) primers for the rapid identification of truncated transcripts in tissue samples of HCC patients
A Hyperimmune Serum against a Synthetic Peptide Corresponding to the Hypervariable Region 1 of Hepatitis C Virus Can Prevent Viral Infection in Cell Cultures

AbstractTo investigate whether a principal neutralization epitope exists in hypervariable region 1 (HVR1) within the putative envelope of hepatitis C virus (HCV), we generated a hyperimmune rabbit serum against a synthetic peptide corresponding to HVR1 of HCV isolate H77. The reactivity of the serum in the enzyme-linked immunosorbent assay was correlated with the 13 amino acids (position 398–410) in HVR1. The serum prevented infection with H77 virus in cell cultures but did not prevent infection with H90 virus, a genetically divergent isolate from the same patient. The study demonstrated that neutralization of HCV was mediated, in part, by isolate-specific antibody recognizing HVR1
Influenza Viruses Display High-Affinity Binding to Human Polyglycosylceramides Represented on a Solid-Phase Assay Surface

AbstractPolyglycosylceramides (PGCs), complex glycolipids containing up to 50 or more sugar residues, are recognized as the minor components of the cell-surface membranes, but a knowledge on their tissue distribution, structure, and function is limited. In this study, the binding of influenza viruses to preparations of PGCs was investigated using a TLC overlay assay and a microwell adsorption assay. The ability of PGCs to bind influenza virus was dependent on the source from which they were derived. Preparations of PGCs from human erythrocytes were found to support binding of A and B influenza virus strains at a much lower concentration than sialyl-6-paragloboside and to be somewhat better receptors for these viruses compared to the sialylglycoprotein fetuin. A high virus-binding activity of PGCs suggests that these species could potentially serve as biologically important cell-surface receptors for influenza viruses
Temperature-Sensitive Mutations in the Protein Kinase-1 (pk-1) Gene of theAutographa californicaNuclear Polyhedrosis Virus That Block Very Late Gene Expression

AbstractWe have developed a genetic screen for temperature-sensitive mutations in the very late transcription apparatus of theAutographa californicanuclear polyhedrosis virus. This method starts with the BacPAK6 virus, which has theEscherichia coli lacZgene under the control of the very late polyhedrin promoter. The desired mutants are temperature-sensitive for β-galactosidase production and can be complemented by wild-type virus, which lacks thelacZgene. Two mutants created by nitrosoguanidine mutagenesis and identified by this screen, and one mutant identified by another screen, have been mapped by marker rescue to the viral protein kinase 1 gene (pk-1). The protein kinase genes of these three mutant viruses have been sequenced, revealing the same point mutation in two of them and a different point mutation in the other. In each case, a single amino acid is changed: In two mutants, XF4 and XF5, Asp 92 is changed to Asn; in the other mutant, KT800, Thr 204 is changed to Ile. Northern blotting of RNA made in cells infected by these three mutant viruses has shown that the accumulation of very late transcripts (lacZandp10) is temperature-sensitive, but that accumulation of at least one late transcript (vp39) is not temperature-sensitive. Nuclear run-on transcription assays with two of the mutants indicate that very late transcription is somewhat temperature-sensitive, although this defect is not as pronounced as the temperature-sensitivity detected by Northern blotting. Transcription of at least one late gene (vp39) is not temperature-sensitive in cells infected by these two mutants. Thus, it appears that the viral protein kinase-1 is involved in very late gene expression. Some of this effect is at the transcription level, but some may also be exerted at the posttranscription level
Long-Term Maintenance of B Cell Immunity to Influenza Virus Hemagglutinin in Mice Following DNA-Based Immunization

AbstractThis study demonstrates that gene-gun inoculation of mice with DNA encoding the influenza virus hemagglutinin (HA) results in the life-long maintenance of protective B cell responses. Using a sensitive single-cell enzyme-linked immunospot assay, we show that all of the HA-specific plasma cells are localized in the bone marrow and spleen 1 year postimmunization. As a consequence of prior virus challenge, only a small population of antibody-forming cells was found in the lymphoid tissues associated with the respiratory tract. The tissue distribution of HA-specific plasma cells in these mice was identical to the profile in infected controls. Complete protection against live virus challenge in the aged vaccinated mice did not require prior exposure to virus. Thus, immunization with the DNA vaccine provides long-term protective immunity against otherwise lethal infection
Induction of Apoptosis by Calmodulin-Dependent Intracellular Ca2+Elevation in CD4+Cells Expressing gp160 of HIV

AbstractHuman CD4+cell clones expressing either gp160 or gp120 of HIV-1 under the transcriptional control of an inducible promoter were used to examine the role of Ca2+signaling in the induction of apoptosis by envelope glycoproteins. Single-cell killing with apoptosis was induced in the cells expressing gp160 while no such apoptosis was found in the cells expressing gp120. An increase of intracellular Ca2+was found in the gp160-expressing cells but not in the gp120-expressing cells as determined by intracellular Ca2+imaging analysis before the appearance of DNA fragmentation. W7, a calmodulin antagonist, blocked the elevation of Ca2+as well as the resultant DNA fragmentation, which thus implies that the calmodulin-dependent intracellular Ca2+release system is first activated by gp160 and thereafter apoptosis takes place. The above results thus indicate that Ca2+signaling plays a crucial role in the apoptosis accompanying the single-cell death induced by gp160 in CD4+cells
Effects of Adenovirus E1A Protein on Interferon-Signaling

AbstractWe have previously shown that adenovirus E1A proteins can block interferon-α (IFN-α)-signaling. In the current study, we examined if the same is true for IFN-γ signaling. Cotransfection experiments showed that both 289R and 243R forms of E1A could block the expression of an IFN-γ-inducible reporter gene. Similarly, in an E1A-expressing HeLa cell line IFN-γ failed to induce the synthesis of IRF-1 mRNA. This failure was due to a block in activation of the crucialtrans-acting factor, GAF, which in turn was due to the lack of IFN-γ-activated tyrosine phosphorylation of the STAT1α protein in E1A-expressing cells. The above defect could be attributed to a reduced level of STAT1α protein. The level of p48 protein, which is required for IFN-α signaling, was also lowered. However, the level of Jak-1 protein, one of the tyrosine kinases necessary for both IFN-α and IFN-γ signaling, was comparable in the E1A-expressing and the control cells. These results indicate that the observed inhibition of IFN signaling in E1A-expressing cells is a consequence of a lower abundance of the necessarytrans-acting factors
Effects of P2 Cleavage Site Mutations on Poliovirus Polyprotein Processing

AbstractThe poliovirus genome comprises a single open reading frame which is translated to give one large polyprotein. The proteolytic cascade involved in the processing of this polyprotein is not yet understood in full detail, particularly concerning the processing of P2–P3, the precursor to the viral nonstructural polypeptides, 2A, 2B, 2C, 3A, 3B, 3C, and 3D. To investigate the possibility that the cleavage events within P2 and at the 2C/3A junction occur in an ordered fashion, we used oligonucleotide-directed mutagenesis of poliovirus cDNA to modify the 3Cpro-mediated cleavage sites. The Gln residue of the Gln-Gly sequence at the 2A/2B, 2B/2C, and 2C/3A junctions in the poliovirus polyprotein was replaced by Asn, Glu, Asp, or Lys. The effects of each of these substitutions were studiedin vivoafter transfection onto HeLa cells andin vitroin a cell-free translation assay, using full-length mutated RNA transcripts. Only the mutant with the Glu-Gly sequence at the 2C/3A junction was viable. Analysis of thein vitroprocessing profiles showed that the efficiency of the 3C protease cleavage at any of the sites in P2 was in the following order: Gln-Gly > Glu-Gly > Asn-Gly. No cleavage could be detected with the Asp-Gly or Lys-Gly sequence at any junction. Lack of 2A/2B or 2B/2C cleavage had no consequences on the cleavage efficiency at other Gln-Gly sites in the polyprotein. Abolition of cleavage at the 2C/3A junction did not prevent the generation of the 2A, 2B, and 3CD polypeptides. Thus, these polypeptides could be produced independently of the generation of the P2 and P3 precursors
Inhibition of Puumala and Tula Hantaviruses in Vero Cells by MxA Protein

AbstractHuman MxA protein is a type I interferon-inducible intracytoplasmic protein, which mediates antiviral actions against a variety of negative-strand RNA viruses including influenza A, measles, and vesicular stomatitis viruses. Recently, it has also been shown that several members of theBunyaviridaefamily are inhibited by MxA protein. The hantavirus genus in theBunyaviridaefamily includes important human pathogenic viruses, e.g., Puumala (PUUV), Hantaan, and Sin Nombre viruses. Tula virus (TULV) is a new member of the genus, but its pathogenicity in man remains to be determined. As assumed by the similarities in replication strategy, MxA would be a good candidate molecule for antiviral action against these viruses, also. To gain more insight into the MxA action on PUUV, we studied PUUV and TULV replication in stably MxA gene-transfected Vero cells. We show that MxA protein has the capacity to inhibit both viral protein and RNA accumulation in virus-infected cells. We also studied PUUV and TULV infection in MxA-transfected U-937 cell clones. In these cell lines both hantaviruses grew poorly, independent of whether the cells were expressing MxA or not. Whether cell line-specific differences in the antiviral activity of MxA protein against hantaviruses exist cannot be conclusively determined due to the lack of productive infection of PUUV and TULV in U-937 cells
Fatal Sindbis Virus Infection of Neonatal Mice in the Absence of Encephalitis

AbstractA comparative pathogenesis study was performed in neonatal mice using a molecularly cloned laboratory variant of Sindbis strain AR339, designated TRSB, and a single-site attenuated mutant of TRSB derived by site-directed mutagenesis of the E2 glycoprotein from Ser to Arg at residue 114 (TRSBr114). TRSB caused 100% mortality with an average survival time of 3.0 ± 0.7 days, whereas mice inoculated with TRSBr114 exhibited an attenuated disease course with 46% mortality and an extended average survival time of 7.5 ± 3.4 days for those animals that died. Reduced virulence of TRSBr114 was characterized by delayed appearance of detectable virus, relative to TRSB, and by lower peak virus titers in both sera and brains of infected mice. TRSB infection induced very high peak serum titers of interferon alpha/beta (215,000 units/ml compared to 2100 units/ml for TRSBr114).In situhybridization analysis demonstrated replication of TRSB in brain, but only minimal histopathological changes and no evidence of encephalitis were observed. However, extensive extraneural lesions and viral replication were found in skin, connective tissue, and muscle. Moreover, dramatic involution of the thymus and loss of hematopoietic tissues were observed in the absence of virus replication at these sites, suggesting the involvement of a systemic physiological stress response in TRSB infection. TRSBr114 infection did not cause thymic lesions. Otherwise, the attenuated mutant demonstrated a similar pattern of tissue and organ involvement, but lesions and positivein situhybridization signal were much more limited in scope and intensity compared to TRSB. TRSBr114-infected mice developed myositis and encephalomyelitis approximately 6 days postinfection. Therefore, TRSB-infected animals may succumb to an early syndrome associated with the stress response, preventing their survival for a time sufficient for the development of encephalitis. Alternatively, a systemic stress response, as evidenced by thymic involution, may result in immunosuppression, thus contributing to the absence of encephalitis. In any event, the attenuating mutation in the E2 glycoprotein significantly altered the course of Sindbis-induced disease by limiting virus replication and associated damage early in infection. Mutant-infected animals survived beyond Day 4 and progressed to a classical encephalomyelitis from which about half recovered
Hepatitis C Virus Core Protein: Carboxy-Terminal Boundaries of Two Processed Species Suggest Cleavage by a Signal Peptide Peptidase

AbstractThe expression and processing of hepatitis C virus core protein was analyzed. Two protein bands, 21 kDa (P21), corresponding to the full-length core, and 19 kDa (P19), were detected as major products when core protein was expressed in the standard rabbit reticulocyte lysate system or inSf9insect cells. Core proteins with amino-terminal hexa-histidine tags were expressed which allowed the purification of the hexa-histidine-P19 core with Ni2+-NTA columns. With the help of mass spectrometry, the molecular weight of hexa-histidine-P19 was analyzed and its carboxy-terminus could be calculated. Fusion proteins of truncated core/core-E1 species fused to mouse dihydrofolate reductase (mDHFR) showed cleavage in the expected region. Cleavage sites could be determined by amino-terminal protein sequencing of the DHFR-fusion partner. Our data show that there are not one but two core products with an apparent molecular weight of about 19 kDa, ending either at amino acid leucine 179 or leucine 182, respectively. These cleavages in the hydrophobic, carboxy-terminal region of HCV core suggest processing by (a) recently proposed eucaryotic signal peptide peptidase(s) (F. Lykoet al.(1995)J. Biol. Chem.270, 19873–19878). Furthermore, our results demonstrate that cleavage at these sites and the formation of the P19 species does not require previous processing at the signalase site (position 191/192) of the HCV-polyprotein
A Murine Host Cell Factor Required for Nicking of the Dimer Bridge of MVM Recognizes Two CG Nucleotides Displaced by 10 Basepairs

AbstractDuring replication of the minute virus of mice (MVM) genome, a dimer replicative form (RF) intermediate is resolved into two monomer RF molecules in such a way as to retain a unique sequence within the left hand hairpin terminus of the viral genome. Although the proposed mechanism for resolution of the dimer RF remains uncertain, it likely involves site-specific nicking of the dimer bridge. The RF contains two double-stranded copies of the viral genome joined by the extended 3′ hairpin. Minor sequence asymmetries within the 3′ hairpin allow the two halves of the dimer bridge to be distinguished. The A half contains the sequenceGAACTT, whereas the B half contains the sequenceTCAG. Using anin vitroassay, we show that only the B half of the MVM dimer bridge is nicked site-specifically when incubated with crude NS-1 protein (expressed in insect cells) and mouse LA9 cellular extract. When highly purified NS-1, the major nonstructural protein of MVM, is used in this nicking reaction, there is an absolute requirement for the LA9 cellular extract, suggesting a cellular factor (or factors) is (are) required. A series of mutations were created in the putative host factor binding region (HFBR) on the B half of the MVM dimer bridge adjacent to the NS-1 binding site. Nicking assays of these B half mutants showed that two CG motifs displaced by 10 nucleotides are important for nicking. Gel mobility shift assays demonstrated that a host factor(s) can bind to the HFBR of the B half of the dimer bridge and efficient binding depends on the presence of both CG motifs. Competitor DNA containing the wild-type HFBR sequence is able to specifically inhibit nicking of the B half, indicating that the host factor(s) bound to the HFBR is(are) essential for site-specific nicking to occur
Isolation and Characterization of Whitewater Arroyo Virus, a Novel North American Arenavirus

AbstractRodents are principal hosts for each of the well-characterized arenaviruses. Prior to the present study, Tamiami (TAM) virus was the sole arenavirus known to be indigenous to North America; it has been isolated only from southern Florida where its primary host is the cotton ratSigmodon hispidus.Recently, arenavirus antibody was found inNeotoma albigulawoodrats collected from the southwestern United States. The purpose of the present study was to isolate and characterize the arenavirus associated withN. albigula.Three isolates of a novel arenavirus (proposed name “Whitewater Arroyo,” WWA) were recovered from two arenavirus antibody-positiveN. albigulacollected from Whitewater Arroyo in McKinley County, New Mexico. Two-way serologic tests indicated that WWA virus is antigenically distinct from other arenaviruses but most closely related to TAM virus. Phylogenetic analysis of nucleocapsid protein gene sequence data showed that WWA virus is a novel arenavirus that is genetically most closely related to TAM virus. The recovery of WWA virus from antibody-positiveN. albigulasuggests that WWA virus infection in this species can be chronic and thus thatN. albigulais a reservoir host of the virus
NP:P and NP:V Interactions of the Paramyxovirus Simian Virus 5 Examined Using a Novel Protein:Protein Capture Assay

AbstractUsing recombinant proteins extracted from mammalian cells, in a novel protein:protein binding assay, direct interaction of the nucleoprotein (NP) of simian virus 5 with the phosphoprotein (P) and V protein (V) was demonstrated. The amount of NP bound by V was found to be significantly less than that bound by P. Furthermore, preabsorption of NP with P removed the fraction of NP that could be bound by V, but preabsorption of NP with V did not remove all the NP that could be bound by P. These results suggested that V bound a subpopulation of the NP recognised by P. Further analysis revealed that P bound both soluble and homopolymeric forms of NP, while V bound only the soluble form; thus demonstrating that the binding sites on P and V, for soluble NP, are located within the N-terminal domain common to both P and V proteins. A monoclonal antibody, which recognised an epitope in the unique C-terminus of P, blocked the binding of P to polymeric NP but not to soluble NP. These results also suggest that there are two binding sites on NP for P; the site that interacts with the P/V common domain being either hidden or conformationally altered in polymeric NP
Resistance to Tomato Yellow Leaf Curl Geminivirus inNicotiana benthamianaPlants Transformed with a Truncated Viral C1 Gene

AbstractThe C1 gene of tomato yellow leaf curl geminivirus (TYLCV) encodes a multifunctional protein (Rep) involved in replication. A truncated form of this gene, capable of expressing the N-terminal 210 amino acids (aa) of the Rep protein, was cloned under the control of the CaMV 35S promoter and introduced intoNicotiana benthamianausingAgrobacterium tumefaciens.The same sequence was also cloned in antisense orientation. When self-pollinated progeny of 19 primary transformants were tested for resistance to TYLCV by agroinoculation, some plants proved to be resistant, particularly in the sense lines. Two such lines were further studied. The presence of the transgene was verified and its expression was followed at intervals. All plants that were resistant to TYLCV at 4 weeks postinoculation (wpi) contained detectable amounts of transgenic mRNA and protein at the time of infection. Resistance was overcome in a few plants at 9 wpi, and in most at 15 wpi. Infection of leaf discs derived from transgenic plants showed that expression of the transgene correlated with a substantial reduction of viral DNA replication. Cotransfections of tobacco protoplasts demonstrated that inhibition of viral DNA replication requires expression of the truncated Rep protein and suggested that the small ORF C4, also present in our construct, plays no role in the resistance observed. The results obtained using both transient and stable gene expression systems show that the expression of the N-terminal 210 aa of the TYLCV Rep protein efficiently interferes with virus infection
Sin Nombre Virus mRNA Synthesis

AbstractSin Nombre (SN) virus is the major etiologic agent of hantavirus pulmonary syndrome, a severe respiratory disease with high mortality. Like other hantaviruses, SN virus causes an inapparent chronic infection of the natural rodent reservoir and tends to grow slowly and produce little cytopathic effect even in highly susceptible Vero E6 tissue culture cells. An electrochemiluminescent quantitative PCR approach was developed to allow examination of SN virus RNA transcription in synchronously infected cells. Although virion particles contain equimolar ratios of the three negative-strand genome RNA segments (S, M, and L), rates and levels of accumulation of the corresponding N, GPC, and L viral mRNAs varied. The smallest mRNA (N) was detectable earliest and plateaued at the highest level, where as the largest mRNA (L) appeared latest and at the lowest plateau level. In addition, all three mRNAs were found to share a common 5′ capped primer initiation mechanism, but appeared to have different mRNA termination mechanisms. The N mRNA 3′ terminus mapped to position 1435 on the S segment, in close proximity to a CCC-rich suspected transcription termination motif. The GPC mRNA 3′ terminus contained a poly(A) tail and mapped to a U8 transcription termination–polyadenylation motif reminiscent of those seen in other negative-strand RNA viruses. Finally, the L mRNA 3′ terminus appeared identical to the L segment antigenome 3′ terminus