pinecone/core-2020-05-10-deduplication huggingface dataset:
Downstream Ribosomal Entry for Translation of Coronavirus TGEV Gene 3b

AbstractGene 3b (ORF 3b) in porcine transmissible gastroenteritis coronavirus (TGEV) encodes a putative nonstructural polypeptide of 27.7 kDa with unknown function that during translation in vitro is capable of becoming a glycosylated integral membrane protein of 31 kDa. In the virulent Miller strain of TGEV, ORF 3b is 5′-terminal on mRNA 3–1 and is presumably translated following 5′ cap-dependent ribosomal entry. For three other strains of TGEV, the virulent British FS772/70 and Taiwanese TFI and avirulent Purdue-116, mRNA species 3–1 is not made and ORF 3b is present as a non-overlapping second ORF on mRNA 3. ORF 3b begins at base 432 on mRNA 3 in Purdue strain. In vitro expression of ORF 3b from Purdue mRNA 3-like transcripts did not fully conform to a predicted leaky scanning pattern, suggesting ribosomes might also be entering internally. With mRNA 3-like transcripts modified to carry large ORFs upstream of ORF 3a, it was demonstrated that ribosomes can reach ORF 3b by entering at a distant downstream site in a manner resembling ribosomal shunting. Deletion analysis failed to identify a postulated internal ribosomal entry structure (IRES) within ORF 3a. The results indicate that an internal entry mechanism, possibly in conjunction with leaky scanning, is used for the expression of ORF 3b from TGEV mRNA 3. One possible consequence of this feature is that ORF 3b might also be expressed from mRNAs 1 and 2
Infectious cDNA Clones of Langat Tick-Borne Flavivirus That Differ from Their Parent in Peripheral Neurovirulence

AbstractTick-borne flavivirus strain Langat TP21 (LGT TP21) recovered from ticks, is naturally attenuated for humans but retains demonstrable neurovirulence and peripheral virulence (“neuroinvasiveness”) for mice. Previously a mutant, strain E5, less virulent for mice was derived from LGT TP21. Multiple attempts to prepare a full-length infectious TP21 cDNA from cDNA fragments cloned in E. coli were uniformly unsuccessful. A more informative sequence than that obtained from these cloned cDNA fragments and similar E5 cDNA fragments was derived from RT-PCR fragments that had not been cloned in E. coli. Comparison of the RT-PCR consensus sequence of TP21 and E5 identified only seven amino acid differences that might be responsible for the observed difference in virulence of these strains for mice. Eleven independent infectious cDNA clones of TP21 were recovered using two overlapping long RT-PCR fragments. Importantly, low-titered virus used to prepare cDNA as template for PCR was harvested early in the growth cycle to minimize the frequency of deletion mutants that accumulated late in infection. The four analyzed rescued clones exhibited clone-specific minimal divergence from the consensus sequence but this limited variation was associated with diminished peripheral virulence for immunocompetent mice. Manipulation of these clones should facilitate elucidation of LGT virulence
Conditional Cell Transformation by Doxycycline-Controlled Expression of the ASV17 v-jun Allele

AbstractTo investigate the molecular basis of oncogenesis induced by the v-jun oncogene of avian sarcoma virus 17 (ASV17), we developed a conditional cell transformation system in which transcription of the ASV17 v-jun allele is controlled by a doxycycline-sensitive transactivator (tTA) or a reverse (doxycycline-dependent) transactivator (rtTA), respectively. Permanent cell lines of quail embryo fibroblasts conditionally transformed by a doxycycline-controlled v-jun allele revert to the normal phenotype within 3 days and lose their ability to grow in soft agar, strictly dependent on the addition or removal of the drug, respectively. The reverted cells are rapidly retransformed on conditional activation of v-jun. While full-level synthesis of v-jun mRNA and v-Jun protein in these cells is established within 2 and 14 h, respectively, after switching to the permissive conditions, the first morphological alterations are observed after 24 h, and as early as 2 days later the morphology has changed entirely from flat cells resembling normal fibroblasts to spindle-shaped fusiform cells showing a typical jun-transformed phenotype. Kinetic expression analysis revealed that transcriptional activation of the direct jun target gene BKJ precisely coincides with the establishment of full-level v-Jun protein synthesis. Furthermore, we have analyzed the expression of a novel candidate v-jun target gene, termed JAC, which shows no sequence homology to known genes. Similar to BKJ, JAC is specifically activated in jun-transformed fibroblasts, and induction of JAC is tightly linked to the conditional expression of oncogenic v-Jun. These results demonstrate the high stringency of the doxycycline-controlled v-jun expression system, and they also indicate that expression of v-jun in these cells is indispensable for enhanced proliferation, cell transformation, and the induction of specific expression patterns of downstream target genes
Precise Mapping of the Replication and Transcription Promoters of Human Parainfluenza Virus Type 3

AbstractThe terminal RNA regions of the genomic and antigenomic RNAs of the paramyxoviruses and rhabdoviruses are known to contain sequences essential for directing RNA replication and transcription. The 3′ terminus (leader region) of the negative-sense, genomic RNA of the rhabdoviruses and paramyxoviruses is known as the leader (Le) promoter and directs synthesis of positive-sense replication and transcription products. The 3′ terminus of the antigenome is termed the trailer complementary (TrC) promoter and directs the synthesis of genomic RNA. By creating mutations in the corresponding regions of an HPIV3 minireplicon in which the viral protein coding sequences were replaced by the luciferase gene, we were able to precisely define the elements of the leader promoter involved in directing positive-strand replication of HPIV3. Nucleotides 1 through 12 (from the terminus) formed a domain critical for replication. The region from nucleotides 13 through 55 was important but not crucial for replication, while G residues at positions 79, 85, and 91 comprised another domain critical for replication. It was also shown that the TrC promoter is similar, though not identical, to the Le promoter. Nucleotides 1 through 12 of the TrC promoter were critical for synthesis of genomic RNA, though specific positions behaved differently from the corresponding positions of the Le promoter. While many of these mutations could not be analyzed for transcription because they completely abrogated genomic RNA synthesis (the template for transcription), we were surprised to find that no mutations in the leader promoter which decreased replication had any significant effect on transcription. However, mutations in the intergenic sequence and gene start signal following the leader and preceding the luciferase message severely decreased transcription, but not replication
Characterization and Immunolocalization of Major Structural Proteins in the Brown Algal Virus EsV-1

AbstractThe Ectocarpus siliculosus virus (EsV-1) is endemic in all populations of the cosmopolitan filamentous brown alga Ectocarpus siliculosus. EsV-1 has a large circular double-stranded DNA genome of about 320 kilobase pairs, and a complex virion structure with a central nucleoprotein core surrounded by several proteinaceous layers. To investigate the protein composition of the virion, we screened an expression library of EsV-1 with antibodies raised against purified detergent-disrupted viral particles. We isolated several clones encoding novel structural proteins and investigated two of them in detail. These clones encode viral proteins vp55 and vp74. Electron microscopy reveals that vp55 is most likely a component of the surface of the viral core, whereas vp74 may be part of an inner core structure. To initiate a genetic analysis, we sequenced regions of the EsV-1 genome encoding vp55 and vp74 and found several adjacent open reading frames with the potential to code for several interesting viral proteins including a putative calcium-binding protein, a collagen-like protein, and a RING finger protein
Homologous Recombination between a Lactococcal Bacteriophage and the Chromosome of Its Host Strain

AbstractGenetic exchanges constitute a significant means by which bacteriophages acquire novel characteristics. Phages of Lactococcus lactis occupy a particular niche, the dairy factory environment, where their populations are subjected to constant changes. Little is known about the mechanisms of evolution that lead to the genetic diversity of lactococcal phages. In this study, we described two DNA exchanges involving the lytic phage ul36, a member of the P335 species, and its L. lactis host. They occurred by homologous recombination with phage-related sequences present in the host chromosome. Both mutants generated by these recombination events are insensitive to the phage resistance mechanism AbiK and one has a reduced burst size as well as a new origin of replication. We propose that this type of DNA exchange with prophages or remnants of prophages occurs frequently within the P335 species as supported by DNA-DNA comparisons between P335-like phages
Monoclonal Antibodies to the Hypervariable Region 1 of Hepatitis C Virus Capture Virus and Inhibit Virus Adsorption to Susceptible Cells in Vitro

AbstractTo analyze the neutralizing-related activity of antibodies against the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) in more detail, monoclonal antibodies (mAbs) against HVR1 were raised by immunizing various strains of mice with one of two synthetic HVR1 peptides that had been derived from two isolates of HCV. The epitope specificity of all six mAbs could be assigned by the use of a series of linear peptides in competitive ELISA. It seems that most subregions in the amino acid sequence of HVR1 can induce a humoral immune response in mice. All three mAbs specific to HVR1-6-1 had the ability to capture homologous HCV-6 and inhibit its absorption to susceptible cells in vitro despite the fact that the epitope of each mAb was at a different location in HVR1, whereas the other three mAbs specific to HVR1-7 could not capture HCV-6 nor inhibit the absorption of HCV-6 to susceptible cells. The data in this study suggest that mAbs against HVR1 can prevent the infectivity of HCV in an isolate-specific and epitope position-independent manner
The Cytoplasmic Tails of the Influenza Virus Spike Glycoproteins Are Required for Normal Genome Packaging

AbstractDeletion of the cytoplasmic tails of the influenza A virus spike glycoproteins, hemagglutinin (HA) and neuraminidase (NA), has previously been shown to result in markedly defective virion morphogenesis (Jin et al., 1997, EMBO J. 16, 1236–1247). We have found that influenza A virus preparations lacking the HA and NA cytoplasmic tails (HAt−/NAt−) have a reduced vRNA to protein content, contain an increase in cellular RNA contaminants, and exhibit increased resistance to ultraviolet (UV) inactivation. There is also a direct correlation between abnormal virion morphology and reduced infectivity. The data suggest that the HAt−/NAt− virion population contains a broader range of number of packaged RNA segments than wild-type (wt) virus. Sucrose gradient centrifugation analysis indicated the presence of a subpopulation of virions with pronounced deformation in virion morphology and reduced infectivity. The role of the HA and NA cytoplasmic tails was examined further by using a trans-complementation assay and it was found that expression of wt HA and NA from cDNAs followed by HAt−/NAt− virus infection caused the formation of a pseudotype virus with wt sedimentation properties. Taken together the data indicate that the HA and NA cytoplasmic tails affect not only virion morphology but also proper genome packaging
Comparison of Two Single-Chain Antibodies That Neutralize Canine Parvovirus: Analysis of an Antibody-Combining Site and Mechanisms of Neutralization

AbstractWe cloned the heavy- and light-chain variable domains of two monoclonal antibodies that recognize each of the two major neutralizing antigenic sites of the canine parvovirus (CPV) capsid. After expression in Escherichia coli as single-chain variable domains (scFv) with glycine-serine linker sequences, both scFv bound CPV capsids with the same specificity as the intact IgG, but with 10- to 20-fold lower avidity. Both scFvs neutralized CPV infectivity with efficiency similar to that of the IgG. Although both IgGs inhibited hemagglutination by CPV, only one scFv was inhibiting. The binding of one of the antibodies has previously been analyzed by cryoelectron microscopic reconstruction and the epitope-binding residues predicted. Mutagenesis of predicted contact residues in three heavy-chain complementarity-determining regions (CDR) showed that mutants of CDR1 or CDR3 reduced the binding of the scFv by about 10-fold compared with the wild-type scFv, while no effect was seen for one mutant of CDR2. The levels of neutralization of CPV and of hemagglutination inhibition by the scFv mutants were proportional to their reduction in binding affinity compared with the wild type. Neither scFv blocked virus binding to host cells, but they both caused aggregation of the capsids and appeared to affect the process of infection after virus uptake into the cells
While E1A Can Facilitate Epithelial Cell Transformation by Several Dominant Oncogenes, the C-Terminus Seems Only to Regulate rac and cdc42 Function, but in Both Epithelial and Fibroblastic Cells

AbstractEpithelial and fibroblast cells were differentially susceptible to transformation by oncogenic src, ras, mos, raf, rac, and cdc42 and the influence of adenovirus E1A. In contrast to NIH 3T3 cells, which are easily transformed by all the oncogenes tested, epithelial cells were more resistant to transformation by the same oncogenes. Transformation efficiency of both primary and immortal epithelial cells by E1B, V12ras, v-src, v-raf, and v-mos was increased by cotransfection of E1A 12S, which enables these cells to overcome the M1/M2 mortality blocks, which are not present in NIH 3T3 cells. NIH 3T3 cell transformation by these oncogenes was not altered by E1A. Although V12cdc42 or V12rac1 alone could produce foci on NIH 3T3 cells, morphological conversion was observed only in the presence of a hypertransforming E1A mutant and not WT E1A. Epithelial cells were not transformed by V12cdc42 or V12rac1, even in the presence of WT or mutant E1A, but could be transformed by coexpression of mos/raf and rac/cdc42, and the resultant phenotype was affected by the E1A C-terminus. Hypertransformation, which has previously been reported with ras and E1A C-terminal mutants, turns out to be due to a synergy with rac/cdc42, but not ERK/MAPK or PI3K ras effectors. Like V12rac, expression of the E1A hypertransforming mutant resulted in the upregulation of vinculin and VASP, concomitant with the altered organization of the actin cytoskeleton in these cells. The results show that in addition to requiring abrogation of M1/M2 mortality blocks, primary epithelial cells require activation of the ERK MAPK cascade and rearrangement of the actin CSK to achieve transformation. In addition, the E1A C-terminus regulates rac/cdc42 function in both epithelial and fibroblast cells to affect the extent of transformation progression
Sendai Virosomes Revisited: Reconstitution with Exogenous Lipids Leads to Potent Vehicles for Gene Transfer

AbstractA reliable new procedure is described for the reconstitution of Sendai viral envelopes suitable for gene transfer. Both fusion and hemagglutinin-neuraminidase glycoproteins were extracted from purified Sendai virus and reconstituted together with DNA in the presence of cholesterol:sphingomyelin:phosphatidylcholine:phosphatidylethanolamine (Chol:SM:PC:PE) in a molar ratio of 3.5:3.5:2:1. Before reconstitution, the DNA to be transferred was condensed by pretreatment with polylysine. Exogenous lipid addition and the DNA-condensation step were essential for maximal size as well as for fusogenic activity of the resulting virosomes, the analysis of which revealed (1) the absence of any genomic material originating from Sendai virus, (2) the presence of fusogenic spikes in a functional orientation, (3) the encapsulation of reporter genes, and (4) high-transfer activity for plasmids carrying the green fluorescent protein (GFP) gene and double-stranded nucleotides into different mammalian cells. Transfer rates were up to 10-fold higher than those obtained with different cationic lipids. Gene delivery by means of our lipid-enriched Sendai virosomes extends the known gene transfer strategies, including those based on Sendai virus previously published
Mutations in Conserved Domains IV and VI of the Large (L) Subunit of the Sendai Virus RNA Polymerase Give a Spectrum of Defective RNA Synthesis Phenotypes

AbstractThe Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein. When aligned with amino acid sequences of L proteins from other negative-sense RNA viruses, the Sendai L protein contains six regions of good conservation, designated domains I–VI, which have been postulated to be important for the various enzymatic activities of the polymerase. To directly address the roles of domains IV and VI, 14 site-directed mutations were constructed either by changing clustered charged amino acids to ala or by substituting selected Sendai L amino acids with the corresponding sequence from measles virus L. Each mutant L protein was tested for its ability to transcribe and replicate the Sendai genome. The series of mutations created a spectrum of phenotypes, from those with significant, near wild-type, activity to those being completely defective for all RNA synthesis. The inactive L proteins, however, were still able to bind P protein and form a polymerase capable of binding the nucleocapsid template. The remainder of the mutations reduced, but did not abolish, enzymatic activity and included one mutant with a specific defect in the synthesis of the leader RNA compared with mRNA, and three mutants that replicated genome RNA much more efficiently in vivo than in vitro. Together, these data suggest that even within a domain, the function of the Sendai L protein is likely to be very complex. In addition, SS3 and SS10 L in domain IV and SS13 L in domain VI were shown to be temperature-sensitive. Both SS3 and SS10 gave significant, although not wild-type, activity at 32°C; however, each was completely inactivated for all RNA synthesis at 37 and 39.6°C. SS13 was completely inactive only when synthesized at the higher temperature. Each polymerase synthesized at 32°C could only be partially heat inactivated in vitro at 39.6°C, suggesting that inactivation involves both thermal lability of the protein and temperature sensitivity for its synthesis
The 23-kDa Protein Coded by the 3′-Terminal Gene of Citrus Tristeza Virus Is an RNA-Binding Protein

AbstractThe 23-kDa protein (p23), encoded by the 3′-proximal gene of the RNA of Citrus tristeza virus (CTV), was overexpressed in Escherichia coli fused to the maltose-binding protein and purified by affinity chromatography. Gel retardation and UV crosslinking assays demonstrated that p23 has the ability to cooperatively bind single-stranded RNA in a non-sequence-specific manner. Formation of the p23-RNA complex was dependent on the conformational state of p23 and on the presence of a basic region, but the complex was stable at high salt concentrations, suggesting that interactions other than those between the negatively charged RNA and the basic region of p23 are involved. Competition assays showed that the affinity of p23 for single-stranded and double-stranded RNA was similar but considerably higher than for single-stranded and double-stranded DNA. By use of a series of artificially generated mutants, the RNA-binding domain of p23 was mapped between positions 50–86, a region containing several basic amino acids and a putative zinc-finger domain. Additional p23-derivatives lacking the conserved residues presumably involved in coordinating the zinc ion showed RNA-binding activity, but with an apparent dissociation constant higher than the wild-type protein. These conserved residues might confer binding specificity or increase binding stability in vivo. Within the Closteroviridae family, p23 is the only protein characterized so far showing RNA-binding activity
An Ovine Adenovirus Vector Lacks Transforming Ability in Cells That Are Transformed by AD5 E1A/B Sequences

AbstractAdenoviruses of the Mastadenovirus and Aviadenovirus genera are able to transform certain cell types and induce tumor formation in susceptible animals. For the mastadenoviruses the E1A/B sequences are largely responsible for these properties but E4 sequences may also be involved. The transforming sequences of the aviadenoviruses, which lack E1A/B and E4 homologues, have not yet been fully identified. The recent proposal for a third genus of adenoviruses, which apparently lack an E1A homologue and have weak E1B homology, prompted an examination of the transforming properties of ovine adenovirus OAV287 (OAV), the prototype member of the new group. When OAV and human adenovirus type 5 (Ad5) were used to infect primary rat embryo cells, transformed foci developed in Ad5- but not in OAV-infected cultures. Similarly, after plasmid transfection, baby rat kidney cells were transformed by Ad5 E1A/B but not by OAV sequences. When CSL503 cells, an ovine cell line that is permissive for OAV, were transfected with Ad5 E1A/B sequences, transformed foci again appeared. However, plasmids or fragments containing complete or partial OAV genome sequences did not detectably transform CSL503 cells under the same conditions. When Ad5 E1A/B sequences were incorporated into the complete OAV genome and transfected, transformed clones were again obtained, showing that the gene dosage and transfection conditions were not limiting for transformation. The provision of Ad5 E1A and OAV sequences in combination marginally increased the number of morphologically altered foci in baby rat kidney cells but failed to induce multilayered focus formation. The data suggest that OAV lacks transforming functions in the cell types examined. Additional information suggesting that OAV may have a fundamentally distinct strategy for replication compared with other Ads is discussed
Rabbit Oral Papillomavirus Complete Genome Sequence and Immunity Following Genital Infection

AbstractRabbit oral papillomavirus (ROPV) infects mucosal tissues of domestic rabbits. The viral genomic sequence has been determined and the most related papillomavirus type was the cutaneous cottontail rabbit papillomavirus (CRPV). Homologies between the open reading frames (ORFs) of ROPV and CRPV, however, ranged from 68% amino acid identity for L1 to only 23% identity for E4. Shared features unique to the two rabbit viruses included a large E6 ORF and a small E8 ORF that overlapped the E6 ORF. Serological responses to ROPV L1 viruslike particles (VLPs) were detected in rabbits infected at either the genital or oral mucosa with ROPV. The antibody response was specific to intact ROPV L1 VLP antigen, was first detected at the time of late regression, and persisted at high levels for several months after complete regression. Both oral and genital lesions regressed spontaneously, accompanied by a heavy infiltrate of lymphocytes. ROPV infection of rabbit genital mucosa is a useful model to study host immunological responses to genital papillomavirus infections