pinecone/core-2020-05-10-deduplication huggingface dataset:
Characterization of the Initiator and Downstream Promoter Elements of Herpes Simplex Virus 1 Late Genes

AbstractPreviously identifiedcis-acting regulatory elements of herpes simplex virus (HSV) 1 late promoters include a TATA element upstream from the start of transcription, an initiator-like element at the start of transcription, and sequences downstream from the start of transcription. To determine whether these elements are functionally equivalent to similar elements from other eukaryotic genes, model late promoters were constructed using well-characterized regulatory elements from non-HSV genes. These modular promoters were then inserted into the viral genome upstream from alacZmarker gene. Results showed that a eukaryotic initiator element, along with a TATA element, can function as a late HSV promoter. Several initiator sequences from both viral and nonviral genes were functionally similar to the initiator-like element in HSV-1 late promoters; however, a random sequence of the same size and a similarly located sequence from the HSV-1 early thymidine kinase promoter could not substitute for the initiator element. These results indicate that eukaryotic initiator elements are functionally equivalent to HSV-1 late promoter initiator elements. In addition, the downstream element of the late glycoprotein C promoter was further analyzed by construction of a series of small deletions and insertions. The presence of the downstream glycoprotein C region in a promoter consisting of a strong TATA and initiator element increased mRNA expression by a modest amount; this effect appeared to be sequence specific and dependent on its exact alignment with the upstream elements of the promoter
Functional Comparisons among Members of the Poxvirus T1/35kDa Family of Soluble CC-Chemokine Inhibitor Glycoproteins

AbstractMany poxviruses express a 35–40-kDa secreted protein, termed “T1” (for leporipoxviruses) or “35kDa” (for orthopoxviruses), that binds CC-chemokines with high affinity but is unrelated to any known cellular proteins. Many previously identified poxvirus cytokine-binding proteins display strict species ligand-binding specificity. Because the T1 and 35kDa proteins share only 40% amino acid identity, we compared the abilities of purified myxoma virus-T1 (M-T1) and vaccinia virus (strain Lister)- and rabbitpox virus-35kDa proteins to inhibit human CC-chemokinesin vitro.All three proteins were equally effective in preventing several human CC-chemokines from binding to target chemokine receptors and blocking subsequent intracellular calcium release. The inhibitory affinities were comparable (Ki= 0.07–1.02 nM). These proteins also displayed similar abilities to inhibit (IC50= 6.3–10.5 nM) human macrophage inflammatory protein-1α-mediated chemotaxis of human monocytes. None of the viral proteins blocked interleukin-8-mediated calcium flux or chemotaxis of human neutrophils, confirming that the biological specificity of the T1/35kDa family is targeted inhibition of CC-chemokines. Despite the significant sequence divergence between the leporipoxvirus T1 and orthopoxvirus 35kDa proteins, our data suggest that their CC-chemokine binding and inhibitory properties appear to be species nonspecific and that the critical motifs most likely reside within the limited regions of conservation
In VitroCharacterization of Late Steps of RNA Recombination in Turnip Crinkle Virus I. Role of the Motif1-Hairpin Structure

AbstractMolecular mechanisms of RNA recombination were studied in turnip crinkle carmovirus (TCV), which has a uniquely high recombination frequency and nonrandom crossover site distribution among the recombining TCV-associated satellite RNAs. Anin vitrosystem has been developed that includes a partially purified TCV replicase preparation (RdRp) and chimeric RNAs that resemble the putativein vivorecombination intermediates (Nagy, P. D., Zhang, C., and Simon, A. E.EMBO J.17, 2392–2403, 1998). This system generates 3′-terminal extension products, which are analogous to the recombination end products. Efficient generation of 3′-terminal extension products depends on the presence of a hairpin structure (termed the motif1-hairpin) that possibly binds to the RdRp. Replacement of the motif1-hairpin with two separate randomized sequences resulted in a basal level of 3′-terminal extension. By using three separate constructs, each carrying similar mutations in the motif1-hairpin, we demonstrate that the role of the motif1-hairpin in 3′-terminal extension is complex and its function is influenced by flanking sequences. In addition to the mutagenesis approach, competition experiments between wild-type and mutated motif1-hairpin constructs suggest that the TCV RdRp likely recognizes the secondary and/or tertiary structure of the motif1-hairpin, while individual nucleotides play a less important role. Overall, the data shed new light into the mechanism of 3′-terminal extension by a viral RdRp that is analogous to the late steps of RNA recombination in TCV
In VitroCharacterization of Late Steps of RNA Recombination in Turnip Crinkle Virus II. The Role of the Priming Stem and Flanking Sequences

AbstractTurnip crinkle carmovirus (TCV) has a uniquely high recombination frequency and nonrandom cross-over site distribution among the recombining TCV-associated satellite RNAs. Anin vitrosystem has been developed that includes a partially purified TCV replicase preparation (RdRp) and chimeric RNAs that resemble the putativein vivorecombination intermediates (Nagy, P. D., Zhang, C., and Simon, A. E.,EMBO J.17, 2392–2403, 1998). This system mimics the strand transfer and primer extension steps of recombination events. We characterize in detail three RNA factors that, in addition to the previously characterized motif1-hairpin, can influence the efficient generation of 3′-terminal extension products: (i) a primer binding region, termed the priming stem; (ii) a spacer region; and (iii) a U-rich sequence located 5′ of the motif1-hairpin. The priming stem is formed between the acceptor RNA and the nascent RNA synthesized from the donor RNA template in the recombinants. The stability and location of the priming stem relative to the motif1-hairpin can influence both the efficiency and initiation site of 3′-terminal extension. A short flexible spacer region between the motif1-hairpin and the priming stem can increase the efficiency of 3′-terminal extensions. A U-rich sequence 5′ of the motif1-hairpin facilitates 3′-terminal extensions and its function partly overlaps with that of the spacer region. These RNA factors may also affect the late steps of RNA recombination in TCV
Echovirus 1 Infection Induces both Stress- and Growth-Activated Mitogen-Activated Protein Kinase Pathways and Regulates the Transcription of Cellular Immediate-Early Genes

AbstractWe have previously shown that echovirus 1 (EV1) infection increases the mRNA levels of cellular immediate-early (IE) genes in host cells. Here we provide further evidence that the induction ofjunB,c-jun,and c-fosgenes is due to active viral macromolecular synthesis rather than to the interaction of EV1 with its receptor, α2β1integrin. Nuclear run-on transcription assays indicated that differences in mRNA levels in infected and uninfected cells are brought about by regulation at the transcriptional level. EV1 infection induced the phosphorylation of both the stress-related p38 mitogen-activated protein kinase (MAPK) and the growth signal-related ERK1/2 MAPKs. Studies with selective MAPK inhibitors revealed that p38 was the main inducer ofjunBexpression, whereas both MAPK pathways were involved in the induction of c-fos.Activation of AP-1 genes was also observed to occur during infections with other enteroviruses and with Semliki Forest A7(74) virus, suggesting that the phosphorylation of MAPKs and induction of AP-1 gene expression may be important regulators of host cell behavior during viral infections
Functional Conservations of the Alkaline Nuclease of Herpes Simplex Type 1 and Human Cytomegalovirus

AbstractThe herpes simplex virus type 1 UL12 gene product, alkaline nuclease (AN), appears to be involved in viral DNA processing and capsid egress from the nucleus (Shao, L., Rapp, L. M., and Weller, S. K.,Virology196, 146–162, 1993). Although the HSV-1 AN is not absolutely essential for viral replication in tissue culture, conservation of the AN gene in all herpesviruses suggests an important role in the life cycle of herpesviruses. The counterpart of HSV-1 AN for human cytomegalovirus (HCMV) is the UL98 gene product. To examine whether the HCMV AN could substitute for HSV-1 AN, we performedtrans-complementation experiments using a HSV-1 amplicon plasmid carrying the HCMV UL98 gene. Our results indicate (i) HCMV AN can complement the growth of the HSV-1 AN deletion mutantUL12lacZvirus intrans;(ii) a new recombinant virus,UL12laZcUL98/99,appears to be generated by the integration of the HCMV UL98 gene into the HSV-1UL12lacZviral genome; (iii) in contrast to its parental HSV-1UL12lacZvirus, capsids formed inUL12lacZUL98/99-infected Vero cells were able to transport from the nucleus to the cytoplasm and mature into infectious viruses. Our results demonstrate a functional conservation of AN between HSV-1 and HCMV
Mushroom Bacilliform Virus RNA: The Initiation of Translation at the 5′ End of the Genome and Identification of the VPg

AbstractMushroom bacilliform virus (MBV) is often found in cultivated mushrooms (Agaricus bisporus) with La France disease. MBV has a 4-kb ssRNA genome of positive-sense encoding four major open reading frames (ORFs). The arrangement of ORFs at the 5′ end of the genome and the deduced amino-acid sequences of two of the putative gene products (protease and RNA-dependent RNA polymerase) show remarkable similarity to some plant viruses, particularly subgroup II luteoviruses. We show that this similarity extends to the translation strategy at the 5′ end of the genome, the presence of a genome-linked protein (VPg), and the location of the VPg downstream of the protease motifs in the polypeptide encoded by ORF2
Neutralization of Different Geographic Strains of the Hepatitis E Virus with Anti-Hepatitis E Virus-Positive Serum Samples Obtained from Different Sources

AbstractA recently developed polymerase chain reaction (PCR)-based cell culture neutralization assay was used to investigate cross-neutralization of known hepatitis E virus (HEV) strains obtained from various HEV-endemic regions of the world with different anti-HEV-positive serum samples. Serum specimens obtained from cynomolgus macaques experimentally infected with strains from Burma, Mexico, or Pakistan cross-neutralized the infectivity of each strain as well as an isolate from Morocco. Serum samples obtained either from infected patients who reside in HEV-endemic regions of the world or from U.S. residents who became infected while traveling to such regions also neutralized all four strains. In contrast, antibodies obtained from rabbits immunized with full-length Burma strain ORF2 protein neutralized only the Burma and Pakistan strains, not the Mexico or Morocco strains. In addition, antibodies obtained from guinea pigs immunized with an N-terminal truncated Burma strain ORF2 protein neutralized each strain except the Morocco strain. These data strongly suggest that antibodies elicited during an HEV infection demonstrate broad HEV neutralizing activity, whereas antibodies elicited after immunization with recombinant Burma ORF2 protein demonstrate a more limited ability to neutralize various HEV strains obtained from different regions of the world endemic for the disease
Apoptosis Precedes Necrosis of Fish Cell Line with Infectious Pancreatic Necrosis Virus Infection

AbstractThe current view of infectious pancreatic necrosis virus (IPNV) infection includes a necrotic process that relies primarily on the histological appearance of tissue after the degenerative process. We tested this view by examining the possibility that apoptosis is a component of double-stranded RNA virus (IPNV) that induces fish embryonic cell death. Four kinds of assays for apoptosis were used in analyzing IPNV-infected CHSE-214 cells: (1) assay with terminal deoxynucleotidyl transferase (TdT)-mediated end-labeling of DNA in nuclei of intact cells during virus infection, (2) assay for procoagulant activity, (3) assay for DNA ladders, and (4) electron microscopic assays for the ultrastructural changes in characteristic apoptotic cells. In all p.i. samples, both low and high m.o.i. groups contained apoptotic nuclei, according to TdT-mediated dUTP labeling of intact cells, but in control CHSE-214 cells, apoptotic nuclei were rare at all levels of incubation sampled by TdT-mediated dUTP labeling. Prenecrotic or postnecrotic cells were found to express phosphatidylserine on the surface by annexin V–FITC labeling, but normal cells did not. DNAs from both 4 h p.i. of high m.o.i. and 8 h p.i. of low m.o.i. were found to be cleaved into fragments indicative of preferential cleavage at internucleosomal sites. The IPNV-infected CHSE-214 cells were analyzed with an electron microscope and showed a pattern of ultrastructural change, indicating that apoptosis appears before pathological changes of necrosis, including condensed chromatin, fragmented nuclei, nuclei with chromatin marginations, and secondary necrosis from prenecrotic cells in IPNV-infected CHSE-214 cells. Together, these findings show that apoptosis precedes any detectable necrotic change in CHSE-214 cells that is currently viewed as necrosis. Thus, apoptosis characterizes the onset of pathology in host cells and is followed by necrotic processes
The 41-kDa Protein of Human Herpesvirus 6 Specifically Binds to Viral DNA Polymerase and Greatly Increases DNA Synthesis

AbstractWe previously isolated a 41-kDa early antigen of human herpesvirus 6 (HHV-6), which exhibited nuclear localization and DNA-binding activity (Agulnicket al.,1993). In this study, we observed that a 110-kDa protein was coimmunoprecipitated with p41 from HHV-6-infected cells by an anti-p41 antibody. This 110-kDa protein was identified as the HHV-6 DNA polymerase (Pol-6) by an antibody raised against the N terminus of Pol-6. Reciprocal immunoprecipitation and Western blot analyses confirmed that p41 complexes with Pol-6 in HHV-6-infected cells. In addition, both p41 and Pol-6 were expressedin vitroand shown to form a specific complex. Anin vitroDNA synthesis assay using primed M13 single-stranded DNA template demonstrated that p41 not only increased the DNA synthesis activity of Pol-6 but also allowed Pol-6 to synthesize DNA products corresponding to full-length M13 template (7249 nucleotides). By contrast, Pol-6 alone could only synthesize DNA of <100 nucleotides. The functional interaction between Pol-6 and p41 appears to be specific because they could not be physically or functionally substitutedin vitroby their herpes simplex virus 1 homologues. Moreover, as revealed by mutational analysis, both the N and C termini of Pol-6 contribute to its binding to p41. In the case of p41, the N terminus is required for increasing DNA synthesis but not binding to Pol-6, whereas the C terminus is totally dispensable
Varicella-Zoster Virus ORF57, Unlike Its Pseudorabies Virus UL3.5 Homolog, Is Dispensable for Viral Replication in Cell Culture

AbstractVaricella zoster virus (VZV) encodes five genes that do not have homologs in herpes simplex virus. One of these genes, VZV ORF57, is predicted to encode a protein containing 71 amino acids. Antibody to ORF57 protein immunoprecipitated a 6-kDa protein in the cytosol of VZV-infected cells. Although the homolog of VZV ORF57 in pseudorabies virus, UL3.5, is critical for viral egress and growth in cell culture, VZV unable to express ORF57 replicated to titers similar to those seen with parental virus. Thus VZV ORF57 has a different role in viral replication than its pseudorabies virus homolog
Virulence Markers in the 5′ Untranslated Region of Genotype 2 Bovine Viral Diarrhea Virus Isolates

AbstractVirulence markers to distinguish high from low virulence bovine viral diarrhea virus genotype 2 isolates have not been previously reported. The objective of this study was to identify virulence markers by evaluating the primary and secondary structures of the 5′-untranslated region of low and high virulence bovine viral diarrhea virus genotype 2 isolates. The nucleotide sequences of the entire 5′-untranslated region mRNA of eight bovine viral diarrhea virus genotype 2 isolates, four of high virulence and four of low virulence, and two genotype 1 reference isolates were determined using a polymerase chain reaction and a 5′ Rapid Amplification of cDNA Ends System. Two nucleotide substitutions were identified in the internal ribosomal entry site that distinguished the high virulence from the low virulence genotype 2 isolates. The low virulence isolates had a cytosine at position 219, whereas the high virulence isolates had a uracil. At position 278, a uracil or cytosine was found in the low and high virulence groups, respectively. The substituted bases are virulence markers that were used to identify bovine viral diarrhea virus genotype 2 isolates of high virulence
Construction and Characterization of E3-Deleted Bovine Adenovirus Type 3 Expressing Full-Length and Truncated Form of Bovine Herpesvirus Type 1 Glycoprotein gD

AbstractUsing the homologous recombination machinery ofE. coli,a 1.245-kb deletion was introduced in the E3 region of bovine adenovirus 3 (BAV3) genomic DNA cloned in a plasmid. Transfection of the restriction enzyme-excised, linear E3-deleted BAV3 genomic DNA into primary fetal bovine retina cells produced infectious virus (BAV3.E3d), suggesting that all the E3-specific open reading frames are nonessential for virus replicationin vitro. Using a similar approach, we constructed replication-competent (BAV3.E3gD and BAV3.E3gDt) BAV3 recombinant expressing full-length (gD) or truncated (gDt) glycoprotein of bovine herpes virus 1. Recombinant gD and gDt proteins expressed by BAV3.E3gD and BAV3.E3gDt, respectively, were recognized by gD-specific monoclonal antibodies directed against conformational epitopes, suggesting that antigenicity of recombinant gD and gDt was similar to that of the native gD expressed in bovine herpes virus 1-infected cells. Intranasal immunization of cotton rats induced strong gD- and BAV3-specific IgA and IgG immune responses. These results suggest that replication-competent bovine adenovirus 3-based vectors have potential for the delivery of vaccine antigens to the mucosal surfaces of animals
Assaying for Structural Variation in the Parvovirus Capsid and Its Role in Infection

AbstractThe capsid of canine parvovirus (CPV) was assayed for susceptibility to proteases and for structural variation. The natural cleavage of VP2 to VP3 in CPV full (DNA containing) particles recovered from tissue culture occurred within the sequence Arg-Asn-Glu-Arg↓Ala-Thr. Trypsin, chymotrypsin, bromelain, and cathepsin B all cleaved >90% of the VP2 to VP3 in full but not in empty capsids and did not digest the capsid further. Digestion with proteinase K, Pronase, papain, or subtilisin cleaved the VP2 to VP3 and also cleaved at additional internal sites, causing particle disintegration and protein degradation. Several partial digestion products produced by proteinase K or subtilisin were ∼31–32.5 kDa, indicating cleavage within loop 3 of the capsid protein as well as other sites. Protease treatment of capsids at pH 5.5 or 7.5 did not significantly alter their susceptibility to digestion. The isoelectric point of CPV empty capsids was pH 5.3, and full capsids were 0.3 pH more acidic, but after proteolysis of VP2 to VP3, the pI of the full capsids became the same as that of the empty capsids. Antibodies against various capsid protein sequences showed the amino termini of most VP2 molecules were on the outside of full but not empty particles, that the VP1-unique sequence was internal, and that the capsid could be disintegrated by heat or urea treatment to expose the internal sequences. Capsids added to cells were localized within the cell cytoplasm in vesicles that appeared to be lysosomes. Microinjected capsids remained primarily in the cytoplasm, although a small proportion was observed to be in the nucleus after 2 h. After CPV capsids labeled with [35S]methionine were bound to cells at 0°C and the cells warmed, little cleavage of VP1 or VP2 was observed even after prolonged incubation. Inoculation of cells with virus in the presence of proteinase inhibitors did not significantly reduce the infection
The Site-Specific Recombination System of theLactobacillusSpecies Bacteriophage A2 Integrates in Gram-Positive and Gram-Negative Bacteria

AbstractThe region of the bacteriophage A2 genome involved in site-specific recombination with the DNA ofLactobacillusspp. has been identified. Twoorfs, transcribed from the same strand, have been found immediately upstream of the phage attachment site (attP). Theorfadjacent toattPpredicts a 385-amino-acid protein that presents significant similarity with site-specific recombinases of the integrase family. The otherorfencodes a basic polypeptide of 76 amino acid residues. The junctions of the prophage with the genomes of its hosts have been determined, allowing the identification of the host attachment site (attB), which has a common 19-nucleotide core region withattP. TheattBsite is located at the 3′ end of the transfer RNALeugene (anticodon CAA). Nonreplicative plasmids containing the A2-specific recombination cassette integrate into different lactobacilli but also into unrelated Gram-positive bacteria such asLactococcus lactisand even intoEscherichia coli. InLc. lactis, integration occurs in a previously unknown intergenic region, whereas inE. coli, it maps within therrnDoperon, 5′ ofrrsDgene. Comparison of the integration sites in the different hosts indicates that some flexibility is permitted in theattBsequence, sinceLc. lactisandE. colionly share 13 and 11 nucleotides, respectively, with the 19-nucleotide core sequence of the lactobacilli
The Paramyxovirus SV5 Small Hydrophobic (SH) Protein Is Not Essential for Virus Growth in Tissue Culture Cells

AbstractThe SH gene of the paramyxovirus SV5 is located between the genes for the glycoproteins, fusion protein (F) and hemagglutinin-neuraminidase (HN), and the SH gene encodes a small 44-residue hydrophobic integral membrane protein (SH). The SH protein is expressed in SV5-infected cells and is oriented in membranes with its N terminus in the cytoplasm. To study the function of the SH protein in the SV5 virus life cycle, the SH gene was deleted from the infectious cDNA clone of the SV5 genome. By using the recently developed reverse genetics system for SV5, it was found that an SH-deleted SV5 (rSV5ΔSH) could be recovered, indicating the SH protein was not essential for virus viability in tissue culture. Analysis of properties of rSV5ΔSH indicated that lack of expression of SH protein did not alter the expression level of the other virus proteins, the subcellular localization of F and HN, or fusion competency as measured by lipid mixing assays and a new content mixing assay that did not require the use of vaccinia virus. The growth rate, infectivity, and plaque size of rSV5 and rSV5ΔSH were found to be very similar. Although SH is shown to be a component of purified virions by immunoblotting, examination of purified rSV5ΔSH by electron microscopy did not show an altered morphology from SV5. Thus in tissue culture cells the lack of the SV5 SH protein does not confer a recognizable phenotype