pinecone/core-2020-05-10-deduplication huggingface dataset:
Varicella-Zoster Virus (VZV) ORF65 Virion Protein Is Dispensable for Replication in Cell Culture and Is Phosphorylated by Casein Kinase II, but Not by the VZV Protein Kinases

AbstractThe unique short region of varicella zoster virus (VZV) encodes four genes. One of these, ORF65, is predicted to encode an 11-kDa protein. Antibody to ORF65 protein immunoprecipitated a 16-kDa protein from the membrane fraction of VZV-infected cells. ORF65 protein was shown to be phosphorylated by casein kinase II. The VZV ORF47 or ORF66 protein kinases were not required for phosphorylation of ORF65. VZV with a large deletion in ORF65 was constructed and was shown to be dispensable for replication of virus in cell culture. The herpes simplex virus homolog of VZV ORF65 has been reported to be located in the nucleus of infected cells and in virions as a tegument protein, whereas the pseudorabies virus homolog is located in the Golgi apparatus of infected cells and in virions as a type II membrane protein. The ORF65 protein localized to the Golgi apparatus in virus-infected cells and was located in virions, most likely as a type II membrane protein. Thus, VZV ORF65 more closely resembles its pseudorabies virus homolog in its localization in infected cells and virions
Kinetic Analyses of Stability of Simple and Complex Retroviral Vectors

AbstractSimple and complex retroviral vectors derived from Moloney murine leukemia virus (MLV) and human immunodeficiency virus type 1 (HIV-1), respectively, are useful tools for gene transfer studies. However, factors affecting the stability of these vectors have not been carefully investigated. Here we studied the stability factors on vesicular stomatitis viral envelope glycoprotein (VSV-G)-pseudotyped MLV- and HIV-1-derived vectors. Analysis of the ratio of defective particles versus infectious units using electron microscopy and a functional transduction assay revealed that both vectors consisted of high numbers of defective particles (∼100–350:1), which could be reduced (∼10–20:1) by centrifugation. Frequent freeze-and-thaw rapidly decreased vector titer in the first three to five cycles and stabilized thereafter. Both viral vectors were sensitive to temperatures above 37°C but more stable at temperatures below 37°C, exhibiting a two-phase inactivation kinetic starting with a steep inactivation phase, followed by a more leveled phase. Interestingly, HIV-1-derived vectors were significantly more stable than MLV-derived vectors at higher temperatures (>37°C). Both vectors were rapidly destabilized at pH either below or above 7.0. Incubation with human or mouse serum significantly inhibited VSV-G-pseudotyped vector activities. Preheated human serum still reduced vector half-lives to ∼50% (150 min), suggesting that certain inactivation factors are not heat-labile. Analyses of these stability factors may improve future production and applications of retroviral and lentiviral vectors
CEVd-Induced Symptom Modification as a Response to a Host-Specific Temperature-Sensitive Reaction

AbstractNatural selection of two new variants of citrus exocortis viroid (CEVd) was detected by observing tissues displaying both severe and mild symptoms from a single Gynura aurantiaca. The variants CEVd-S (severe) and CEVd-M (mild), differing by only five nucleotides confined to the pathogenic (P) domain, remained stable when propagated by rooted cuttings or from successive plants inoculated with tissue extracts or transcripts from cDNA clones. CEVd-S induces a very severe reaction in Gynura that is consistent throughout a range of environmental conditions. However, symptoms resulting from CEVd-M infection can vary from a nonsymptomatic condition to a severe reaction when grown at 40°C. This differential response was confined to a single host, Gynura aurantiaca, and expressed under standard growing conditions. The distinct host responses induced by these variants could not be correlated with any changes in sequence or conformation of the dominant viroid variant, as predicted by molecular modeling. Therefore, the variable symptom expression appears to be associated with a specific temperature-sensitive response of Gynura aurantiaca
Glycoprotein D Homologs in Herpes Simplex Virus Type 1, Pseudorabies Virus, and Bovine Herpes Virus Type 1 Bind Directly to Human HveC (Nectin-1) with Different Affinities

AbstractDistinct subsets of human receptors for alphaherpesviruses mediate the entry of herpes simplex virus (HSV), pseudorabies virus (PrV), or bovine herpes virus type 1 (BHV-1) into cells. Glycoprotein D (gD) is essential for receptor-mediated entry of all three viruses into cells. However, the gD homologs of these viruses share only 22–33% amino acid identity. Several entry receptors for HSV have been identified. Two of these, HveA (HVEM) and HveC (nectin-1), mediate entry of most HSV-1 and HSV-2 strains and are bound directly by HSV gD. A third receptor, HveB (nectin-2), mediates entry of HSV-2 and only a limited number of HSV-1 strains. HveB and HveC can also serve as entry receptors for PrV, whereas only HveC can serve this function for BHV-1. We show here that gD from PrV and BHV-1 binds directly to the human receptors that mediate PrV and BHV-1 entry. We expressed soluble forms of PrV gD and BHV-1 gD using recombinant baculoviruses and purified each protein. Using ELISA, we detected direct binding of PrV gD to HveB and HveC and direct binding of BHV-1 gD to HveC. Biosensor analysis revealed that PrV gD had a 10-fold higher affinity than HSV-1 gD for human HveC. In contrast, the binding of BHV-1 gD to HveC was weak. PrV gD and HSV-1 gD competed for binding to the V domain of HveC and both inhibited entry of the homologous and heterologous viruses. These data suggest that the two forms of gD bind to a common region on human HveC despite their low amino acid similarity. Based on affinities for human HveC, we predict a porcine HveC homolog may be important for PrV infection in its natural host, whereas a BHV-1 infection in its natural host may be mediated by a receptor other than a bovine HveC homolog
Interaction between the J3R Subunit of Vaccinia Virus Poly(A) Polymerase and the H4L Subunit of the Viral RNA Polymerase

AbstractJ3R, the 39-kDa subunit of vaccinia virus poly(A) polymerase, is a multifunctional protein that catalyzes (nucleoside-2′-O-)-methyltransferase activity, serves as a poly(A) polymerase stimulatory factor, and acts as a postreplicative positive transcription elongation factor. Prior results support an association between poly(A) polymerase and the virion RNA polymerase. A possible direct interaction between J3R and H4L subunit of virion RNA polymerase was evaluated. J3R was shown to specifically bind to H4L amino acids 235–256, C terminal to NPH I binding site on H4L. H4L binds to the C-terminal region of J3R between amino acids 169 and 333. The presence of a J3R binding site near to the NPH I binding region on H4L led us to evaluate a physical interaction between NPH I and J3R. The NPH I binding site was located on J3R between amino acids 169 and 249, and J3R was shown to bind to NPH I between amino acids 457 and 524. To evaluate a role for J3R in early gene mRNA synthesis, transcription termination, and/or release, a transcription-competent extract prepared from cells infected with mutant virus lacking J3R, J3-7. Analysis of transcription activity demonstrated that J3R is not required for early mRNA synthesis and is not an essential factor in early gene transcription termination or transcript release in vitro. J3R interaction with NPH I and H4L may serve as a docking site for J3R on the virion RNA polymerase, linking transcription to mRNA cap formation and poly(A) addition
Identification of an Initiator-like Element within the HTLV-I Promoter

AbstractUsing the 5′ long terminal repeat (LTR) as its only promoter, the HTLV-1 provirus generates a single RNA transcript that undergoes differential splicing to express the various viral proteins. Examination of sequence near the transcription start site revealed an element resembling a transcriptional initiator (Inr) at position −8 to −15 in addition to the canonical TATA box at −25. To elucidate basal control of HTLV-I gene expression, functional traits of this element were examined. It specifically bound a protein complex, the mobility of which was altered by antibody to serum response factor, and independently mediated reporter gene expression. Mutating the Inr in a minimal construct reduced basal transcription, whereas mutation of the element within the context of the complete LTR left basal transcription unaffected. Presence of the element influenced transcription start site choices. Exhibiting many characteristics of an Inr, this element may play an important role in regulating HTLV-I gene expression in vivo, particularly during the long clinical latency period prior to development of HTLV-I-induced disease
Alterations in HIV-1 Rev Transport in Response to Cell Stress

AbstractMovement of HIV-1 Rev between the nucleus and cytoplasm is essential to its function. While normally nuclear, the protein can be induced to accumulate in the cytoplasm upon inhibition of RNA polymerase I/II. Nuclear accumulation of Rev in the presence of these inhibitors was found to be rescued upon addition of leptomycin B, an inhibitor of Rev nuclear export. This finding, in conjunction with kinetic data on nuclear import, indicates that the effect of the RNA polymerase inhibitors is due to an inversion of the rates of nuclear import versus export possibly achieved by increasing the rate of Rev nuclear export. We also examined whether changes in Rev localization could be due to a stress response. While neither ultraviolet radiation nor heat shock affected Rev subcellular localization, both oxidative and osmotic shocks induce changes in Rev localization comparable to that observed with the RNA polymerase inhibitors. The ability of certain serine/threonine kinase inhibitors, including CKI/II inhibitors, to cause cytoplasmic accumulation of Rev suggested that the alteration in Rev distribution could be due to changes in Rev or CRM1 phosphorylation. However, no change in extent of phosphorylation of either protein is observed upon treatment of cells with any of the agents tested, indicating involvement of another cellular factor
Inhibition of R5X4 Dualtropic HIV-1 Primary Isolates by Single Chemokine Co-receptor Ligands

AbstractThe susceptibility of HIV-1 to chemokine-mediated inhibition may be lost as a consequence of the expanded usage of chemokine co-receptors frequently occurring in clade B isolates obtained from individuals with advanced disease. Since chemokine-based immune intervention is under intense investigation, it is crucial to determine its potential effect on primary dualtropic HIV isolates characterized by simultaneous utilization of CCR5 and CXCR4 chemokine co-receptors (R5X4 viruses). In the present study, the CCR5 binding chemokine regulated upon activation normal T cell expressed and secreted (RANTES) strongly inhibited the replication of two of eight primary R5X4 viruses in mitogen-activated primary peripheral blood mononuclear cells (PBMC). The CXCR4 antagonist AMD3100 efficiently suppressed the replication of other two HIV isolates, whereas the remaining four viruses were partially inhibited by treatment with either RANTES or AMD3100. The potency of chemokine-mediated inhibition was influenced by PBMC donor variability, but it was usually independent from the levels of expression of CCR5 or CXCR4. Dual co-receptor usage was maintained by the viruses after two serial passages on U87.CD4 astrocytic cell lines expressing exclusively either CCR5 or CXCR4. The gp120 env variable domains were sequenced before and after passages on U87.CD4 cells. Virus replication into U87.CD4-CXCR4 cells did not result in changes in the V3 region but perturbed the dominant env V4 sequence. Interestingly, double passage onto U87.CD4-CXCR4 cells determined the loss of susceptibility to RANTES inhibition. In conclusion, interference with CCR5 may efficiently inhibit the replication of at least some dualtropic HIV-1 strains, whereas forced CXCR4 usage may result in viral escape from CCR5-dependent inhibitory effects
Immune Response to Vaccinia Virus Recombinants Expressing Glycoproteins gE, gB, gH, and gL of Varicella-Zoster Virus

AbstractImmunogenicity of Varicella-zoster virus glycoproteins gE, gB, gH, and gL expressed by recombinant vaccinia viruses (VV) separately or simultaneously was determined in mice and guinea pigs by ELISA, Western blotting, radioimmunoprecipitation, plaque reduction assay, and skin test. Single VV-gE and VV-gB recombinants and double VV-gH/gL recombinant elicited specific antibodies with VZV neutralizing activity in mice. Co-expression of gE and gB by one recombinant VV resulted in an increased antibody response in comparison with immunization with single recombinants or their mixtures. Unlike anti-gB and anti-gH/gL antibodies, the gE-specific antibodies had no virus neutralizing activity in absence of complement, and when used alone, they even caused considerable increase of VZV infectious units. Moreover, immune sera containing anti-gE antibodies antagonized complement independent virus-neutralizing activity of anti-gB- and anti-gH/gL-positive sera. The ability to induce delayed hypersensitivity reaction to VZV antigens was observed after immunization of guinea pigs with gE- and/or gB-expressing VVs
Three Membrane-Proximal Amino Acids in the Human Parainfluenza Type 2 (HPIV 2) F Protein Are Critical for Fusogenic Activity

AbstractIn this study, we investigated the role of the membrane-proximal region of the human parainfluenza virus type 2 (HPIV2) F protein by mutational analysis, including deletion, insertion, and substitution. Deletion or replacement of the entire 12 amino acid region (aa 474–485) of the HPIV2 F protein completely abolished its fusion activity when coexpressed with the HPIV2 HN protein. Deletion of groups of four of aa 478–485, single alanine, or other amino acid substitutions among aa 478–485 had minimal or limited effects on HPIV2 F/HN-induced cell fusion. However, a significant reduction in, or complete inhibition of, fusion activity was observed when aa 474–477 were deleted, or the N475, F476, or F477 residues were singly substituted with alanine. In addition, insertions of four amino acids at this region or deletion of eight or more amino acids significantly reduced F protein fusion activity. The oligomerization patterns and levels of cell surface expression of the mutant F proteins were compared to those of the wild-type HPIV2 F protein. The mutant HPIV2 F proteins defective in fusion were also found to be unable to initiate hemifusion, indicating that there is a specific requirement for three specific amino acids as well as the spacing in this region for initiating lipid mixing
The Efficacy of Potent Anti-retroviral Drug Combinations Tested in a Murine Model of HIV-1 Encephalitis

AbstractDevelopment of anti-retroviral regimens with enhanced efficacy against brain HIV-1 is essential if viral eradication is to be achieved. To address this, a severe combined immune deficiency mouse model of HIV-1 encephalitis was used to assay the effect of protease-containing and protease-sparing drug regimens on viral replication in brain macrophages. Here, HIV-1-infected human monocyte-derived macrophages (MDM) are inoculated into basal ganglia, causing a multinucleated giant cell encephalitis reminiscent of human disease. Drugs were administered at the time of MDM inoculation and continued until sacrifice. Immunohistochemical tests evaluated ongoing viral replication, glial immunity, and neuronal survival. Treatment with ddI/d4T decreased the numbers of infected cells by 75%, while ddI/d4T/amprenavir or ZDV/3TC/ABC diminished infection by 98%. Triple drug regimens decreased astrogliosis by ≥25%. This small-animal model may be used to screen drug regimens that affect ongoing HIV-1 replication within its brain sanctuary
Amplification of Citrus Tristeza Virus from a cDNA Clone and Infection of Citrus Trees

AbstractIsolates of the Closterovirus, Citrus tristeza virus (CTV), are populations of disparate genotypes and defective RNAs developed during long periods of vegetative propagation of citrus trees. Because it has not been possible to obtain pure cultures of the virus, it is not known what components of the population are primarily responsible for induction of diseases. We previously developed an infectious cDNA clone from which in vitro-produced RNA transcripts could infect protoplasts (Satyanarayana et al., 1999, Proc. Natl. Acad. Sci. USA 96, 7433–7438). However, neither the RNA transcripts nor virions from transcript-infected protoplasts were competent for infection of citrus trees. Using a green fluorescent protein-marked virus as inoculum, we found that the ∼20-kb RNA from virions or transcripts of cDNA infected only a small percentage of protoplasts (∼0.01%), but virions could infect more than 80% of the protoplasts. Based on this information, we amplified the virus from the cDNA clone (recombinant virus) by successive passages in protoplasts using virions in crude sap as inoculum. By the third to seventh passages in protoplasts, maximal amounts of recombinant progeny virus were produced, which were used for inoculation of small citrus trees by slashing stems in the presence of virion preparations. A relatively high percentage of plants became infected with the recombinant virus from protoplasts, resulting in the first defined pure culture of CTV in plants. The comparative biology of the pure culture of recombinant CTV with that of the parental population in planta demonstrated that the recombinant virus retained through all of the recombinant DNA manipulations the normal functions of replication, movement, and aphid transmissibility, and had a symptom phenotype indistinguishable from that of the parental population. Additionally, fulfilling Koch's postulates of the first pure culture of CTV in plants suggested that the major genotype of the CTV T36 population is the primary determinant of the symptom phenotype. We could distinguish no biological contributions resulting from the minor genotypes and defective RNAs of the parental population
Activation of Human Rhinovirus-14 3C Protease

AbstractThe catalytic efficiency of human rhinovirus-14 (HRV14) 3C protease as a function of solvents and other regulators has been investigated using synthetic peptides as substrates. The proteolytic activity of HRV14 3C was found to be strongly stimulated by a series of anions in vitro and the activation was accompanied by changed Km, kcat, and increased kcat/Km values. A more than 72-fold increase in the 3C catalytic efficiency toward peptide substrates was observed in the presence of 0.8 M sodium sulfate. Several approaches, including size-exclusion chromatography and chemical cross-linking experiments, suggested that no oligomerization of the 3C enzyme occurred in the presence of activating anions. However, the anions did induce a significant conformational change of HRV14 3C protease, as revealed by circular dichroism spectrometry and tyrosine fluorescence analyses, which might contribute to 3C enzyme activation. Finally, the results obtained from 3C protease inhibitor studies suggested that the S1 specificity pocket of HRV14 3C was mainly affected by the activating anions. An induced-fit catalysis mechanism for viral proteases is discussed
The Genome Sequence of Yaba-like Disease Virus, a Yatapoxvirus

AbstractThe genome sequence of Yaba-like disease virus (YLDV), an unclassified member of the yatapoxvirus genus, has been determined. Excluding the terminal hairpin loops, the YLDV genome is 144,575 bp in length and contains inverted terminal repeats (ITRs) of 1883 bp. Within 20 nucleotides of the termini, there is a sequence that is conserved in other poxviruses and is required for the resolution of concatemeric replicative DNA intermediates. The nucleotide composition of the genome is 73% A+T, but the ITRs are only 63% A+T. The genome contains 151 tightly packed open reading frames (ORFs) that either are ≥180 nucleotides in length or are conserved in other poxviruses. ORFs within 23 kb of each end are transcribed toward the termini, whereas ORFs within the central region of the genome are encoded on either DNA strand. In the central region ORFs have a conserved position, orientation, and sequence compared with vaccinia virus ORFs and encode many enzymes, transcription factors, or structural proteins. In contrast, ORFs near the termini are more divergent and in seven cases are without counterparts in other poxviruses. The YLDV genome encodes several predicted immunomodulators; examples include two proteins with similarity to CC chemokine receptors and predicted secreted proteins with similarity to MHC class I antigen, OX-2, interleukin-10/mda-7, poxvirus growth factor, serpins, and a type I interferon-binding protein. Phylogenic analyses indicated that YLDV is very closely related to yaba monkey tumor virus, but outside the yatapoxvirus genus YLDV is more closely related to swinepox virus and leporipoxviruses than to other chordopoxvirus genera
Protection of Rabbits against Rabbit Hemorrhagic Disease Virus by Immunization with the VP60 Protein Expressed in Plants with a Potyvirus-Based Vector

AbstractA new plum pox potyvirus (PPV)-based vector has been constructed for the expression of full-length individual foreign proteins. The foreign sequences are cloned between the NIb replicase and capsid protein (CP) cistrons. The heterologous protein is split from the rest of the potyviral polyprotein by cleavage at the site that originally separated the NIb and CP proteins and at an additional NIa protease recognition site engineered at its amino-terminal end. This vector (PPV–NK) has been used to clone different genes, engendering stable chimeras with practical applications. We have constructed a chimera expressing high levels of jellyfish green fluorescent protein, which can be very useful for the study of PPV molecular biology. The VP60 structural protein of rabbit hemorrhagic disease virus (RHDV) was also successfully expressed by making use of the PPV–NK vector. Inoculation of extracts from VP60-expressing plants induced a remarkable immune response against RHDV in rabbits, its natural host. Moreover, these animals were protected against a lethal challenge with RHDV
Resistance to Fas-Mediated Apoptosis of Human T-Cell Lines Expressing Human T-Lymphotropic Virus Type-2 (HTLV-2) Tax Protein

AbstractThe susceptibility to Fas-mediated apoptosis was evaluated in seven T-cell lines (two infected with HTLV-2, one with HTLV-1, and four HTLV-free) as well as in Jurkat cells transfected with a Tax-2 expressing vector. Fas-mediated apoptosis was significantly reduced in the HTLV-1- and HTLV-2-infected lines in comparison with the HTLV-free lines regardless of the surface expression of Fas antigen (which was no different in the infected and uninfected cells). Fas-mediated apoptosis was also significantly inhibited in Jurkat cells transfected with the Tax-2 expressing vector without any modification in Fas expression. There was significantly more antiapoptotic Bcl-xL mRNA and protein in the transfected than in the untransfected Jurkat T cells. In conclusion, our results suggest that HTLV-2 is capable of inhibiting Fas-mediated apoptosis by means of a mechanism involving the tax-2 gene and probably the expression of bcl-xL messenger and protein