pinecone/core-2020-05-10-deduplication huggingface dataset:
Live and Killed Rhabdovirus-Based Vectors as Potential Hepatitis C Vaccines

AbstractA highly attenuated, recombinant rabies virus (RV) vaccine strain-based vector was utilized as a new immunization strategy to induce humoral and cellular responses against hepatitis C (HCV) glycoprotein E2. We showed previously that RV-based vectors are able to induce strong immune responses against human immunodeficiency virus type I (HIV-1) antigens. Here we constructed and characterized three replication-competent RV-based vectors expressing either both HCV envelope proteins E1 and E2 or a modified version of E2 which lacks 85 amino acids of its carboxy terminus and contains the human CD4 transmembrane domain and the CD4 or RV glycoprotein cytoplasmic domain. All three constructs stably expressed the respective protein(s) as indicated by Western blotting and immunostaining. Moreover, surface expression of HCV E2 resulted in efficient incorporation of the HCV envelope protein regardless of the presence of the RV G cytoplasmic domain, which was described previously as a requirement for incorporation of foreign glycoproteins into RV particles. Killed and purified RV virions containing HCV E2 were highly immunogenic in mice and also proved useful as a diagnostic tool, as indicated by a specific reaction with sera from HCV-infected patients. In addition, RV vaccine vehicles were able to induce cellular responses against HCV E2. These results further suggest that recombinant RVs are potentially useful vaccine vectors against important human viral diseases
Targeted Therapy of Respiratory Syncytial Virus in African Green Monkeys by Intranasally Administered 2-5A Antisense

AbstractRespiratory syncytial virus (RSV) is a leading cause of respiratory disease in infants, young children, immunocompromised patients, and the institutionalized elderly. Previous work had shown that RNase L, an antiviral enzyme of the interferon system, could be recruited to cleave RSV genomic RNA by attaching tetrameric 2′-5′-linked oligoadenylates (2-5A) to an oligonucleotide complementary to repetitive gene-start sequences within the RSV genome (2-5A antisense). A 2′-O-methyl RNA-modified analog of the lead 2-5A anti-RSV chimera is shown here to have enhanced antiviral activity in cell culture studies while also cleaving RSV genomic RNA in an RNase L- and sequence-specific manner. When administered intranasally to RSV-infected African green monkeys, this chimera reduced nasal RSV replication by up to four log10 units in a dose- and time-dependent manner
Poliovirus 3C Protease-Mediated Degradation of Transcriptional Activator p53 Requires a Cellular Activity

AbstractInfection of HeLa cells with poliovirus leads to rapid shut-off of host cell transcription by RNA polymerase II. Previous results have suggested that both the basal transcription factor TBP (TATA-binding protein) and transcription activator proteins such as CREB (cyclic AMP-responsive element-binding protein) and Oct-1 (the octamer-binding factor) are cleaved by the viral-encoded protease, 3CPro. Here we demonstrate that the transcriptional activator (and tumor suppressor) p53 is degraded by the viral protease 3C both in vivo and in vitro. Unlike other transcription factors that are directly cleaved by 3Cpro, degradation of p53 requires a HeLa cell activity in addition to 3CPro. The degradation of p53 by 3CPro does not appear to involve the ubiquitin pathway of protein degradation. Vaccinia virus infection of HeLa cells leads to inactivation of the cellular activity required for 3CPro-mediated degradation of p53. The vaccinia-encoded protein (CrmA) is known to inhibit caspase I (ICE protease) that converts inactive IL-1β to an active secreted form. Incubation of HeLa cells with caspase I inhibitor Z-VAD-fmk does not interfere with 3CPro-mediated degradation of p53. The cellular activity present in extracts of HeLa cells can be fractionated through phosphocellulose. A partially purified fraction that elutes at 0.6 M KCl from phosphocellulose contains the activity that degrades p53 in a 3CPro-dependent manner. These results suggest that both poliovirus-encoded protease 3CPro and a cellular activity are required for the degradation of p53 observed in cells infected with poliovirus
Involvement of RNA2-Encoded Proteins in the Specific Transmission of Grapevine Fanleaf Virus by Its Nematode Vector Xiphinema index

AbstractThe nepovirus Grapevine fanleaf virus (GFLV) is specifically transmitted by the nematode Xiphinema index. To identify the RNA2-encoded proteins involved in X. index-mediated spread of GFLV, chimeric RNA2 constructs were engineered by replacing the 2A, 2BMP, and/or 2CCP sequences of GFLV with their counterparts in Arabis mosaic virus (ArMV), a closely related nepovirus which is transmitted by Xiphinema diversicaudatum but not by X. index. Among the recombinant viruses obtained from transcripts of GFLV RNA1 and chimeric RNA2, only those which contained the 2CCP gene (504 aa) and 2BMP contiguous 9 C-terminal residues of GFLV were transmitted by X. index as efficiently as natural and synthetic wild-type GFLV, regardless of the origin of the 2A and 2BMP genes. As expected, ArMV was not transmitted probably because it is not retained by X. index. These results indicate that the determinants responsible for the specific spread of GFLV by X. index are located within the 513 C-terminal residues of the polyprotein encoded by RNA2
The Small 11-kDa Protein from B19 Parvovirus Binds Growth Factor Receptor-Binding Protein 2 in Vitro in a Src Homology 3 Domain/Ligand-Dependent Manner

AbstractThe small 11-kDa proteins of B19 parvovirus contain three proline-rich regions which conform to consensus Src homology 3 (SH3) ligand sequences present in signaling molecules within the cell. We have shown that the B19 11-kDa proteins specifically interact with the growth factor receptor-binding protein 2 (Grb2) in vitro. Mutation of prolines within one of the three SH3 ligand-like sequences decreases the binding of B19 11-kDa proteins to Grb2, suggesting that the proline-rich region is involved in the B19 11-kDa/Grb2 interaction. Therefore, the B19 11-kDa proteins may function to alter Grb2-mediated signaling by disrupting SH3 domain/ligand interactions. These results implicate the 11-kDa proteins in B19 pathogenesis through perturbation of normal cellular signaling pathways
Analysis of Early Region 1 of Porcine Adenovirus Type 3

AbstractTo identify the proteins encoded by the porcine adenovirus 3 (PAV-3) E1 region, rabbit antisera were prepared using a bacterial fusion protein encoding E1A, E1Bsmall, or E1Blarge protein. Sera against E1A, E1Bsmall, and E1Blarge immunoprecipitated a protein of 35, 23, and 53 kDa, respectively, in in vitro translated and transcribed mRNA and PAV-3 infected cells. To determine the role of E1 proteins in PAV-3 replication, we constructed vectors with a deletion(s) in the E1 region. Mutant PAV211, containing deletions in E1A and E3, grew to titers similar to wild-type in VIDO R1 cells (E1A complementing) but not in swine testicular (ST) cells. No early protein (E1Bsmall, DNA binding protein) expression could be detected in PAV211 infected ST cells by Western blots. Mutant PAV212, containing deletions in E1Bsmall and E3, grew to wild-type titers in VIDO R1 or ST cells. These deletions were successfully rescued, resulting in recombinant PAV214, containing deletions in E1A, E1Bsmall, and E3. However, mutant PAV-3, containing a triple stop codon inserted in the E1Blarge coding sequence, could not be isolated. Next, we constructed a recombinant PAV216 by inserting the green fluorescent protein gene flanked by a promoter and a poly(A) in the E1A region of the PAV214 genome. Both PAV214 and PAV216 replicate as efficiently as wild-type in VIDO R1 cells. These results suggested that (a) E1A is essential for virus replication and is required for the activation of other PAV-3 early genes, (b) E1Bsmall is not essential for replication of PAV-3, and (c) E1Blarge is essential for virus replication. Moreover, the PAV216 vector can be used for the expression of a transgene
Hybrid Papillomavirus L1 Molecules Assemble into Virus-like Particles That Reconstitute Conformational Epitopes and Induce Neutralizing Antibodies to Distinct HPV Types

AbstractHuman papillomavirus (HPV) hybrid virus-like particles (VLPs) were prepared using complementary regions of the major capsid L1 proteins of HPV-11 and -16. These hybrid L1 proteins were tested for assembly into VLPs, for presentation and mapping of conformational neutralizing epitopes, and as immunogens in rabbits and mice. Two small noncontiguous hypervariable regions of HPV-16 L1, when replaced into the HPV-11 L1 backbone, produced an assembly-positive hybrid L1 which was recognized by the type-specific, conformationally dependent HPV-16 neutralizing monoclonal antibody (N-MAb) H16.V5. Several new N-MAbs that were generated following immunization of mice with wild-type HPV-16 L1 VLPs also recognized this reconstructed VLP, demonstrating that these two hypervariable regions collectively constituted an immunodominant epitope. When a set of hybrid VLPs was tested as immunogens in rabbits, antibodies to both HPV-11 and -16 wild-type L1 VLPs were obtained. One of the hybrid VLPs containing hypervariable FG and HI loops of HPV-16 L1 replaced into an HPV-11 L1 background provoked neutralizing activity against both HPV-11 and HPV-16. In addition, conformationally dependent and type-specific MAbs to both HPV-11 and HPV-16 L1 VLP were obtained from mice immunized with hybrid L1 VLPs. These data indicated that hybrid L1 proteins can be constructed that retain VLP-assembly properties, retain type-specific conformational neutralizing epitopes, can map noncontiguous regions of L1 which constitute type-specific conformational neutralizing epitopes recognized by N-MAbs, and trigger polyclonal antibodies which can neutralize antigenically unrelated HPV types
Rotavirus Cross-Species Pathogenicity: Molecular Characterization of a Bovine Rotavirus Pathogenic for Pigs

AbstractRotaviruses which cause disease in heterologous animal species have been reported but the molecular basis of cross-species infectivity and disease is not established. We report the molecular characterization of a cloned rotavirus, PP-1, which was originally obtained from cattle and which had been biologically characterized in vivo in two target animal species, gnotobiotic pigs and calves. In pigs, PP-1 caused severe clinical disease but in experimental calves it replicated subclinically. PP-1 was characterized as a G3 reassortant with a porcine VP4 and NSP4 but a bovine NSP1. The PP-1 VP4 had 96 to 97% deduced amino acid identity to P[7] porcine rotaviruses and P[7] specificity was confirmed with VP4-specific monoclonal antibodies. Sequence analysis of the PP-1 NSP1 showed 94 to 99.6% deduced amino acid identity to bovine rotaviruses but the NSP4 protein had 94 to 98% identity to the NSP4 genotype B porcine rotaviruses. G-typing PCR initially classified PP-1 as a G10 rotavirus but sequence analysis revealed 92 to 96% identity of the PP-1 VP7 with porcine, simian, and human G3 rotaviruses. These results, combined with the in vivo properties of PP-1 in the two target species, supported the concept that species-specific VP4 and NSP4, but not NSP1, are required to induce rotavirus disease, at least in calves and pigs. The results illustrate experimentally that rotaviruses circulating in one animal species can pose a risk to another by the emergence of a pathogenic reassortant rotavirus under appropriate conditions
Marek's Disease Virus VP22: Subcellular Localization and Characterization of Carboxyl Terminal Deletion Mutations

AbstractMarek's disease virus (MDV) is an alphaherpesvirus that causes T cell lymphoma and severe immunosuppression in chickens. The MDV UL49 gene, which encodes the tegument viral protein 22 (VP22), has been expressed as a green fluorescent protein (GFP) fusion protein in chicken embryonic fibroblasts to examine its subcellular localization. As with both human herpesvirus 1 and bovine herpesvirus 1VP22-GFP fusion proteins, the MDV VP22-GFP product binds to microtubules and heterochromatin. In addition, the MDV protein also binds to the centrosomes. During mitosis, VP22-GFP binds to sister chromatids, but dissociates from the centrosomes and the microtubules of the mitotic spindle. A series of VP22 carboxy terminal truncation mutants were constructed to define regions responsible for these binding properties. These mutants identified separable domains or motifs responsible for binding microtubules and heterochromatin
Immunolocalization Studies of an Antisense Protein in HIV-1-Infected Cells and Viral Particles

AbstractThe minus DNA strand of HIV-1 presents an open reading frame that is complementary to the HIV-1 envelope messenger, is highly conserved among HIV-1 isolates, and may encode a hydrophobic protein. In previous studies, the antisense transcript has been identified both in various HIV-infected cell lines and in leukocytes of HIV-1+ patients. The expression of the corresponding antisense protein (ASP) during natural HIV-1 infection has been indirectly evidenced by the identification of anti-ASP antibodies in the sera of HIV+ patients. We have used immunoelectron microscopy procedures (ultrasmall gold particles coupled to silver enhancement) to establish direct evidence of ASP production. ASP has then been detected in chronically and acutely HIV-1-infected cell lines. The protein, found mostly associated with various cellular membranes as well as with the virions released from the cells, indicated that ASP is a bona fide component for the virions and may participate in the particle formation
Hepatitis C Virus NS5A Colocalizes with the Core Protein on Lipid Droplets and Interacts with Apolipoproteins

AbstractThe nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) has been shown to interact with a variety of cellular proteins and implicated in the regulation of cell growth, interferon resistance, and other cellular signaling pathways, but the role of NS5A in HCV pathogenesis has not been firmly established. To further characterize this multifunctional protein, we instigated the studies of the subcellular localization of NS5A in a hepatoma cell line. NS5A was localized to the perinuclear membrane structures, including the endoplasmic reticulum (ER) and the Golgi apparatus, by immunofluorescence staining and confocal microscopy. In addition, it was also associated with the surface of cytoplasmic globular structures when expressed alone or as a part of the NS3-5B polyprotein. Oil red O staining revealed that these globular structures were lipid droplets, where the HCV core protein was also localized. The association of NS5A with intracellular membrane was further confirmed by membrane flotation analysis. To determine whether NS5A interacts with any cellular lipid-binding protein, we performed yeast two-hybrid screening in both HepG2 and human liver cDNA libraries. Apolipoprotein A1 (apoA1), one of the protein components of high-density lipoprotein (HDL) particles, was identified by two independent screening processes. The interaction between NS5A and apoA1 was confirmed by both in vitro pull-down and in vivo coimmunoprecipitation experiments. Immunofluorescence staining revealed a significant colocalization of NS5A and apoA1 in the Golgi apparatus. Our results established an association of NS5A with lipid droplets and apoA1, suggesting that NS5A, together with the core protein, may play a role in the pathogenesis of the derangement of lipid metabolism, contributing to liver steatosis commonly observed in hepatitis C
Hepatitis C Virus Core Protein Induces Cell Proliferation and Activates ERK, JNK, and p38 MAP Kinases Together with the MAP Kinase Phosphatase MKP-1 in a HepG2 Tet-Off Cell Line

AbstractHepatitis C virus (HCV) core protein is a multifunctional protein interacting with cellular and viral proteins and promoters. A tetracycline-regulated system was used to generate a HepG2 Tet-Off cell line allowing regulated expression of a full-length (191 aa) and an Nc-truncated core protein (160 aa). In this system HCV core protein expression activates extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 mitogen-activated protein (MAP) kinase, induces MAP kinase phosphatase MKP-1 expression, and increases cell proliferation. This was accompanied by an activation of c-Jun and ATF-2, but not Elk-1 and c-Fos. Furthermore, AP-1 activation was independent of c-Fos. Full-length and Nc-truncated HCV core proteins exerted similar effects
Out-of-Frame versus In-Frame Core Internal Deletion Variants of Human and Woodchuck Hepatitis B Viruses

AbstractHuman hepatitis B virus (HBV) variants containing in-frame core internal deletion (CID) have been demonstrated to contain all the functional features of defective interfering (DI) particles (Yuan, T. T.-T., M.-H. Lin, D. S. Chen, and C. Shih, 1998, J. Virol. 72, 578–584). Here, we report that out-of-frame HBV CID variants exhibit defective interfering property similar to in-frame CID variants characterized previously. This result raises the possibility that it may be the deleted pregenomic RNA product, rather than the deleted core protein product, that is responsible for interference. Furthermore, a genomic deletion elsewhere does not cause interference since preS2 deletion variants exhibit no influence on wild-type HBV replication. Consistent with the natural occurrence of HBV CID variants, we recently identified CID variants of woodchuck hepatitis virus (WHV) in natural infection. However, unlike HBV CID variants, functional characterization of WHV CID variants using a human hepatoma cell line has not revealed any interference in tissue culture. In summary, defective interference is a general phenomenon for both in-frame and out-of-frame HBV CID variants
Restriction of a Sindbis Virus Mutant in BHK Cells and Relief of the Restriction by the Addition of Adenosine

AbstractSVPZF is a mutant of Sindbis virus (SV) which we selected on the basis of its ability to replicate in mosquito cells treated with pyrazofurin (PZF), a drug which inhibits pyrimidine nucleotide biosynthesis (Lin et al., 2000, Virology 272, 61–71). Three mutations, A6627U, A7543U, and C7593A, were identified in the nsP4 (the viral RNA polymerase) coding region, which were required for the PZF-resistant phenotype. We report here that SVPZF has a second phenotype. Its replication in BHK cells is severely restricted; yields of SVPZF from BHK cells are 100- to 1000-fold lower than the yields of standard SV (SVSTD). However, addition of adenosine to the SVPZF-infected cultures completely relieves this restriction and results in yields comparable to those observed with SVSTD. Adenosine has no effect on the yield of SVSTD from BHK cells. Synthesis of the viral structural proteins is markedly depressed in SVPZF-infected BHK cells, as is synthesis of the viral subgenomic (SG) RNA from which these proteins are translated. In contrast, normal amounts of genomic RNA are made. Experiments with mutagenized viruses indicated that the SVPZF mutation, C7593A, by itself, was sufficient to produce the restriction phenotype. However, this mutation not only changes Pro 609 of nsP4 to Thr, it also changes the nucleotide at the −5 position of the SG promoter. To evaluate the relative contributions of the change in nsP4 and the change in the SG promoter to the restriction phenotype, we made use of double SG viruses, in which nsP4 and the promoter for the SG RNA which encodes the structural proteins can be changed independent of each other. Our results indicated that both the change in nsP4 and the change in the SG promoter were required to produce the full restriction phenotype. We suggest that the changes in nsP4 and the SG promoter destabilize the RNA initiation complex assembled at the SG promoter and that since ATP is the initiating nucleotide in the SG RNA transcript, the increased level of ATP resulting from the addition of adenosine is able to compensate for this destabilization and restore the synthesis of SG RNA to normal levels
A New Cellular Factor Recognizes E2 Binding Sites of Papillomaviruses Which Mediate Transcriptional Repression by E2

AbstractRepression of transcription by the full-length E2 protein of papillomaviruses (PV) seems to occur when the E2 binding sites and those of positively acting cellular factors overlap. Previously, we showed that RUNX1 (formerly called CBF) binds to the repression-mediating E2 binding site P2 of human PV type 8 (HPV8). By a yeast one-hybrid system we could identify an unknown protein binding also to P2, tentatively called PBF (papillomavirus binding factor). PBF recognizes the sequence CCGG, which represents the 3′ half of the E2 binding site just adjacent to the RUNX1 motif. PBF also binds to the repression-mediating E2 BS-1 in BPV1, which is conserved to P2 of HPV8. Point mutations destroying PBF binding to HPV8 P2 and BPV-1 E2 BS-1 in vitro reduce promoter activity in corresponding reporter constructs. Our results suggest that PBF might play a role in transcription of PV genes and in E2-mediated repression
Heparan Sulfate-Mediated Binding of Infectious Dengue Virus Type 2 and Yellow Fever Virus

AbstractDengue virus type 2 and Yellow fever virus are arthropod-borne flaviviruses causing hemorrhagic fever in humans. Identification of virus receptors is important in understanding flavivirus pathogenesis. The aim of this work was to study the role of cellular heparan sulfate in the adsorption of infectious Yellow fever and Dengue type 2 viruses. Virus attachment was assessed by adsorbing virus to cells, washing unbound virus away, releasing cell-bound virus by freezing/thawing, and then titrating the released infectious virus. Treatment of cells by heparin-lyase, desulfation of cellular heparan sulfate, or treatment of the virus with heparin inhibited cell binding of both viruses. Heparin also inhibited Yellow fever virus infection by 97%. Using infectious virus, the present work shows the importance of heparan sulfate in binding and infection of these two flaviviruses
Vaccinia Virus CrmE Encodes a Soluble and Cell Surface Tumor Necrosis Factor Receptor That Contributes to Virus Virulence

AbstractPoxviruses encode soluble cytokine receptors to interfere with host immune functions. Cells infected with vaccinia virus (VV) strains USSR, Lister, and Evans express soluble and cell surface tumor necrosis factor receptors (vTNFRs). We have characterized vTNFR activity in VV USSR and identified an open reading frame that encodes both soluble and cell surface activity, hereafter referred to as VV cytokine response modifier E (VV CrmE). Expression and characterization from recombinant VV and baculovirus showed VV CrmE to be an 18-kDa protein that bound human, mouse, and rat TNF-α, but not human LTα. VV CrmE inhibited the cytotoxic and apoptotic activities of human, but not mouse or rat, TNF in vitro. Nonetheless, in a murine intranasal model, USSR recombinants lacking CrmE were attenuated, demonstrating a role in vivo. Furthermore, expression of VV or cowpox virus vTNFRs from VV strain WR (which itself does not express a vTNFR) was shown to enhance virulence in the murine model