pinecone/core-2020-05-10-deduplication huggingface dataset:
Foot-and-Mouth Disease Virus Lacking the VP1 G-H Loop: The Mutant Spectrum Uncovers Interactions among Antigenic Sites for Fitness Gain

AbstractThe Arg-Gly-Asp (RGD) triplet found in the G-H loop of capsid protein VP1 of foot-and-mouth disease virus (FMDV) is critically involved in the interaction of FMDV with integrin receptors and with neutralizing antibodies. Multiplication of FMDV C-S8c1 in baby hamster kidney 21 (BHK-21) cells selected variant viruses exploiting alternative mechanisms of cell recognition that rendered the RGD integrin-binding triplet dispensable for infectivity. By constructing chimeric viruses, we show that dispensability of the RGD in these variant FMDVs can be extended to surrounding amino acid residues. Replacement of eight amino acid residues within the G-H loop of VP1 by an unrelated FLAG marker yielded infectious virus. Evolution of FLAG-containing viruses in BHK-21 cells generated complex quasispecies in which individual mutants included amino acid replacements at other antigenic sites of FMDV. Inclusion of such replacements in the parental FLAG clone resulted in an increase of relative fitness of the viruses. These results suggest structural or functional connections between antigenic sites of FMDV and underscore the value of mutant spectrum analysis for the identification of fitness-promoting genetic modifications in viral populations. The possibility of producing viable viruses lacking antigenic site A may find application in the design of new anti-FMD vaccines
Heterogeneous Distribution of TT Virus of Distinct Genotypes in Multiple Tissues from Infected Humans

AbstractTT virus (TTV) DNA was quantitated in the serum and nine autopsy tissues (bone marrow, lymph node, muscle, thyroid gland, lung, liver, spleen, pancreas, and kidney) obtained from each of three TTV-infected subjects by real-time polymerase chain reaction (PCR), which can detect all TTV genotypes. TTV DNA was detected in all examined tissues, with the viral load being equal to or up to 300 times higher than that in the corresponding serum (2.1 × 105 to 5.3 × 107 copies/g vs 1.2–3.9 × 105 copies/ml). Generally, the TTV viral load was higher in the bone marrow, lung, spleen, and liver than in the other tissues, although it varied by individual. Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified TTV DNA of 3.3 kilobases (kb) revealed considerable differences among the TTVs in the serum and tissue specimens from each subject. Further, the 3.3-kb amplicons from the serum and tissue specimens from one subject were molecularly cloned, and 30 clones each from the serum and each tissue specimen were subjected to RFLP and sequence analysis (total, 300 clones): the TTV clones were classified into six genotypes including four novel genotypes. The genotypic variability was remarkable: each specimen had one to five TTV genotypes at different frequencies. TTV DNA in replicative intermediate forms and TTV mRNA were detectable in all tissues tested. These results indicate the broad, uneven distribution of TTV genotypes in tissues and suggest that viral replication takes place in multiple tissues at distinct levels in infected individuals
Further Identification and Characterization of Novel Intermediate and Mature Cleavage Products Released from the ORF 1b Region of the Avian Coronavirus Infectious Bronchitis Virus 1a/1b Polyprotein

AbstractThe coronavirus 3C-like proteinase is one of the viral proteinases responsible for processing of the 1a and 1a/1b polyproteins to multiple mature products. In cells infected with avian coronavirus infectious bronchitis virus (IBV), three proteins of 100, 39, and 35 kDa, respectively, were previously identified as mature cleavage products released from the 1b region of the 1a/1b polyprotein by the 3C-like proteinase. In this report, we show the identification of two more cleavage products of 68 and 58 kDa released from the same region of the polyprotein. In addition, two stable intermediate cleavage products with molecular masses of 160 and 132 kDa, respectively, were identified in IBV-infected cells. The 160-kDa protein was shown to be an intermediate cleavage product covering the 100- and 68-kDa proteins, and the 132-kDa protein to be an intermediate cleavage product covering the 58-, 39-, and 35-kDa proteins. Immunofluorescent staining of IBV-infected cells and cells expressing individual cleavage products showed that the 100-, 68-, and 58-kDa proteins were associated with the membranes of the endoplasmic reticulum, and the 39- and 35-kDa proteins displayed diffuse distribution patterns
In Vivo and in Vitro Characterization of an RNA Replication Enhancer in a Satellite RNA Associated with Turnip crinkle virus

AbstractRNA replication enhancers are cis-acting elements that can stimulate replication or transcription of RNA viruses. Turnip crinkle virus (TCV) and satC, a parasitic RNA associated with TCV infections, contain stem-loop structures that are RNA replication enhancers (P. Nagy, J. Pogany, and A. E. Simon, EMBO J. 1999, 18, 5653–5665). We have found that replacement of 28 nt of the satC enhancer, termed the motif1-hairpin, with 28 randomized bases reduced satC accumulation 8- to 13-fold in Arabidopsis thaliana protoplasts. Deletion of single-stranded flanking sequences at either side of the hairpin also affected RNA accumulation with combined alterations at both sides of the hairpin showing the most detrimental effect in protoplasts. In vitro analysis with a partially purified TCV RdRp preparation demonstrated that the motif1-hairpin in its minus-sense orientation was able to stimulate RNA synthesis from the satC hairpin promoter (located at the 3′ end of plus strands) by almost twofold. This level of RNA synthesis stimulation is ∼fivefold lower than that observed with a linear promoter, suggesting that a highly stable hairpin promoter is less responsive to the presence of the motif1-hairpin enhancer than a linear promoter. The motif1-hairpin in its plus-sense orientation was only 60% as active in enhancing transcription from the hairpin promoter. Since the motif1-hairpin is a hotspot for RNA recombination during plus-strand synthesis and since satC promoters located on the minus-strand are all short linear sequences, these findings support the hypothesis that the motif1-hairpin is primarily involved in enhancing plus-strand synthesis
Hepatitis C Virus Core Protein Modulates the Interferon-Induced Transacting Factors of Jak/Stat Signaling Pathway But Does Not Affect the Activation of Downstream IRF-1 or 561 Gene

AbstractHepatitis C virus (HCV) has a propensity to cause chronic infection, with a low proportion of patients exhibiting a sustained response to interferon-α (IFNα) therapy. An earlier report suggested that HCV inhibits IFNα-induced signal transduction through the Jak/Stat pathway by preventing the formation of the transacting factor ISGF3 complex, although the effect on downstream pathway and the specific viral protein responsible for inhibition of IFNα-mediated signal transduction were not elucidated. HCV core protein displays a number of intriguing functional properties and has been implicated in virus-mediated pathogenesis. In this study, we have analyzed the effect of core protein upon IFNα- or IFNγ-induced regulation of the Jak/Stat signaling pathway. HCV core protein expression exhibited a reduced Stat1 expression in IFN-treated mammalian cells. A gel retardation assay suggested a reduced level of formation of the transacting factors, GAF and ISGF3, in IFN-treated cells. Further studies from protein expression and RNase protection assay revealed that the reduced level of GAF or ISGF3 formation could be attributed to modulation of Stat1 protein expression, an important player for innate immunity in host defense mechanism. However, these modulatory effects did not interfere with the activation of the downstream effector genes, IRF-1 and 561, in IFN-treated cells. Stable transfectants of cells after introduction of a plasmid DNA encoding both the structural and the nonstructural proteins of HCV also exhibited a similar effect. Taken together, these results suggest that although expression of the core protein alone or with other HCV proteins modulate transacting factors of Jak/Stat signaling pathway, expression of the downstream effector genes IRF-1 and 561 remains unaffected upon IFN treatment and may contribute to host defense mechanism
Association of L* Protein of Theiler's Murine Encephalomyelitis Virus with Microtubules in Infected Cells

AbstractWe used an antibody raised against a synthetic peptide corresponding to amino acid residues 70–88 for characterizing the L* protein of Theiler's murine encephalomyelitis virus (TMEV), which is only synthesized in DA subgroup strains from an alternative AUG and is out of frame with the viral polyprotein; evidence suggests that L* protein is critical to viral persistence, demyelination, and growth in murine macrophage cell lines. It was synthesized with kinetics similar to that of other viral proteins, although less in amount. After synthesis, it remained stable in the cytoplasm and was not incorporated into virions. Immunofluorescent staining and immunoblotting of microtubule preparations demonstrated that it is associated with microtubules. Expression of L* protein also demonstrated that the 5′ one third of the coding region may be responsible for the association. The association of L* protein with microtubules may be important in the disease-inducing and in vitro characters of L* protein
A Molecular Clock Dates the Common Ancestor of European-type Porcine Reproductive and Respiratory Syndrome Virus at More Than 10 Years before the Emergence of Disease

AbstractThe disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) emerged independently and almost simultaneously in Europe (1990) and North America (1987). The original reservoir of the virus and the date it entered the pig populations is not known. In this study, we demonstrate an accurate molecular clock for the European PRRSV ORF 3 gene, place the root in the genealogy, estimate the rate of nucleotide substitution, and date the most recent common viral ancestor of the data set to 1979; more than 10 years before the onset of the European epidemic. Based on these findings, we conclude that PRRSV virus most likely entered the pig population some time before the epidemic emergence of the virus, and hence, that emergence of European-type PRRSV is not the result of a recent species transmission event. Together, our results show that ORF3 sequencing is a valuable epidemiologic tool for examining the emergence and spread of PRRSV in Europe. As such, the panel of well-characterized and highly divergent ORF3 sequences described in this study provides a reference point for future molecular epidemiologic studies
Intranuclear Localization of the Transcription Coadaptor CBP/p300 and the Transcription Factor RBP-Jk in Relation to EBNA-2 and -5 in B Lymphocytes

AbstractWe have studied the expression and the localization of the cellular proteins CBP/p300 and RBP-Jk in in vitro EBV-infected human B lymphocytes in relation to the EBNA-2 and EBNA-5 proteins. We found that the level of CBP/p300 was elevated drastically by EBV infection and also after activation by CD40 ligation. Thus the increase in CBP/p300 expression in the EBV-infected cells is related to the virus-induced activation and proliferation of the cells. EBNA-2 and RBP-Jk colocalized in the nucleoplasm, which is in accordance with their functional interaction. We confirmed earlier reports about the presence and colocalization of EBNA-5 and CBP in the nuclear POD bodies. On the other hand, neither EBNA-2 nor p300 was detected in the PODs. The expression of these two proteins overlapped in some distinct dots of the nucleoplasm. Taken together, the different patterns of CBP and p300 expression and their different localization in relation to the PML bodies and two EBV-encoded proteins in the B cells may provide some clue to their distinct functional roles
Bovine Adenovirus Type 3 E1Bsmall Protein Is Essential for Growth in Bovine Fibroblast Cells

AbstractIn order to study the function of bovine adenovirus type 3 (BAV-3) E1A and E1Bsmall proteins, we constructed two mutants: (a) BAV102A carries an in-frame deletion in the coding region for the E1A protein (nt 831–1080); (b) BAV102B carries an insertion of triple stop codons in the E1B region (nt 1654, 178 bp downstream of the E1Bsmall start codon), which stops the translation of the E1Bsmall gene. BAV102A virus could grow to the wild-type BAV-3 titer in transformed cell line VIDO R2 (HAV-5 E1 transformed) cells, but no progeny virus could be found in fetal bovine retina cells (FBRC). RT–PCR and Western blot analysis showed that neither mRNA transcripts nor protein expression of early genes [E1Bsmall and DNA binding protein (DBP)] could be detected in BAV102A infected FBRC. The BAV102B grew 1.5 log less than wild-type BAV-3 in FBRC; however, no BAV102B progeny virus could be observed in bovine fibroblast (BFB) cells. No appreciable difference was observed in DBP transcript synthesis between wild-type BAV-3- or BAV102B-infected FBRC. However, compared to wild-type BAV-3, BAV102B viral DNA synthesis and fiber gene expression were found to be slightly reduced in FBRC. In contrast, compared to wild-type BAV-3, DBP transcripts and viral DNA synthesis were drastically reduced in BAV102B-infected BFB cells. In addition, no fiber gene expression could be detected in BAV102B-infected BFB cells. These results suggest that BAV-3 E1A is essential for virus replication and is required for activating the transcription of other BAV-3 early genes. However, the requirement for E1Bsmall protein for BAV-3 replication appears to be cell type-dependent
Synergistic Effects of Gene-End Signal Mutations and the M2-1 Protein on Transcription Termination by Respiratory Syncytial Virus

AbstractIndividual mononegavirus genes terminate with a short cis-acting element, the gene-end (GE) signal, that directs polyadenylation and termination and might also influence the efficiency of reinitiation at the next downstream gene. The 12–13 nucleotide (nt) GE signals of human respiratory syncytial virus (RSV) consist of a conserved pentanucleotide (3′-UCAAU, negative sense), followed by a 3-nt middle region that is AU-rich but otherwise not conserved, followed by a 4- or 5-nt poly(U) region that is thought to generate the poly(A) tail of the encoded mRNA by reiterative copying. Most of the naturally occurring differences in the GE signals of the various RSV genes occur in the “middle” and “poly(U)” regions. We mutated a copy of the fusion protein (F) GE signal that was positioned at the end of the promoter-proximal gene of a tricistronic minigenome and evaluated the effect of these mutations on RSV transcription in a plasmid-initiated, intracellular assay. Mutations confirmed the importance of the middle region's AU-rich nature and 3-nt length, and the poly(U) tract's 4-nt minimum functional length, with maximal termination efficiency observed at five U residues. Nt assignments other than U at position 13 also affected the efficiency of termination, showing that this position is part of the functional 13-nt GE signal. These results indicate that differences in nt assignments in the middle and poly(U) regions of the GE signal, which occur frequently in nature, affect the efficiency of termination. Unexpectedly, the ability of certain mutations to inhibit termination was completely dependent on coexpression of the M2-1 protein, and in many other cases the inhibitory effect of the mutation was greatly enhanced in the presence of M2-1. Thus, M2-1 appears to have the effect of altering the polymerase such that it ignores suboptimal GE signals. Interestingly, certain mutations that greatly decreased the efficiency of termination in the absence of M2-1 did not have much effect on the expression of the second gene, implying that correct termination and/or polyadenylation at the upstream gene is not obligatory for reinitiation at the next downstream gene
A Single Amino Acid Substitution in the Viral Polymerase Creates a Temperature-Sensitive and Attenuated Recombinant Bovine Parainfluenza Virus Type 3

AbstractBovine parainfluenza virus type 3 (bPIV3) is under development as a live virus vaccine vector. The RNA genome of a recombinant bPIV3 harbored four nucleotide changes, one of which resulted in a mutation of the viral polymerase (A. A. Haller et al., 2000, J. Virol. 74, 11626–11635). The contribution of this conservative amino acid substitution (I1103V) in the polymerase to the temperature-sensitive and attenuation phenotypes of r-bPIV3 was investigated by creating a new virus, r-bPIV3(I), that expressed the wild-type polymerase. r-bPIV3(I) was not temperature-sensitive for growth in vitro and the replication of r-bPIV3(I) was no longer restricted in hamsters. The effect of the amino acid substitution in the polymerase was also studied in a chimeric bovine/human PIV3, a virus that displayed temperature-sensitive and attenuated phenotypes (A. A. Haller et al., 2000, J. Virol. 74, 11626–11635). It was not clear whether these defects were due to the impaired polymerase or the replacement of the bPIV3 surface glycoproteins with those of hPIV3. The results showed that the altered polymerase was indeed responsible for the temperature-sensitive phenotype of bovine/human PIV3 but did not appear to play a role in the attenuation phenotype
Functional Analysis of the Influenza A Virus cRNA Promoter and Construction of an Ambisense Transcription System

AbstractWhile influenza A viral RNA is known to act as a template for the synthesis of both viral mRNA and complementary cRNA, the latter has been observed so far only to function as an intermediate in replication and give rise to progeny vRNA molecules. Here it is shown that the cRNA promoter is also capable of initiating viral mRNA synthesis, similar to vRNA-promoted transcription adhering to the cap-snatching mode of primer recruitment. Detection of cRNA promoted transcription required an inversion of the reporter gene coding sequence plus relocation of the viral polyadenylation signal. Construction of cRNA promoter variants through RNA polymerase I reverse genetics allowed us to determine the RNA polymerase-associated, base-paired conformation in a reporter gene read-out system. It again turned out to adhere to the “corkscrew” model, similar, but slightly different in its binding interactions from the corresponding vRNA conformation. The observation of two transcription reactions, initiated in either direction from influenza vRNA and cRNA template molecules, allowed us to construct bicistronic, ambisense RNA molecules for simultaneous expression of two proteins from a single segment of viral RNA
Role of the Glu Residues of the Influenza Hemagglutinin Fusion Peptide in the pH Dependence of Fusion Activity

AbstractTo elucidate the role of the fusion peptide in influenza hemagglutinin (HA)-mediated fusion, we compared pH-dependent conformational changes and fusion mediated by wild-type and a mutant HA in which Glu residues at positions 11 and 15 of the fusion peptide are substituted for valine. The pH dependence of conformational changes and kinetics of fusion with erythrocytes was the same for both forms of HA. The time for commitment and the temperature dependence of HA-mediated fusion were also the same. However, striking differences were observed between wild-type and mutant fusion peptides in their interactions with lipid membranes at neutral and acidic pH. Since elimination of the negatively charged residues allows the exposed fusion peptide to penetrate the bilayer at pH values closer to neutral, but does not affect conformational changes and fusion activity in intact HA, we conclude that conformational changes are tightly coupled to fusion peptide insertion in the overall HA-mediated fusion cascade
Virus Targeting of the Tumor Necrosis Factor Superfamily

AbstractHerpesviruses appear to peacefully coexist with their natural hosts, with infection typically manifested as a benign, but lifelong process. However, coexistence depends on active resistance by innate and specific immune defenses as revealed in the striking virulence of herpesviruses when immunity fails. This pattern of infection is characteristic of a viral pathogen, such as cytomegalovirus, that has evolved efficient strategies targeted at host defense systems. Targeting members of the tumor necrosis factor (TNF)/lymphotoxin (LT) superfamily of cytokines is a strategy found in all herpesviruses, which suggests the existence of an intimate evolutionary link in their host–parasite relationship. Here we examine some of the strategies used by herpesvirus that target members of the TNF superfamily and discuss a recent study that revealed a novel mechanism that links LT-related ligands and interferons (IFN) to the establishment of coexistence between herpesvirus and its host cell
Green Fluorescent Protein Expression from Recombinant Lettuce Infectious Yellows Virus-Defective RNAs Originating from RNA 2

AbstractLettuce infectious yellows virus (LIYV) RNA 2 defective RNAs (D RNAs) were compared in protoplasts for their ability to replicate and to express the green fluorescent protein (GFP) from recombinant D RNA constructs. Initially four LIYV D RNAs of different genetic composition were compared, but only two (LIYV D RNA M5 and M18) replicated to high levels. Both of these contained at least two complete ORFs, one being the 3′-terminal ORF encoding P26. Northern hybridization analysis using probes corresponding to 3′ regions of LIYV RNA 2 detected the P26 subgenomic RNA from protoplasts infected with LIYV RNAs 1 and 2 or protoplasts inoculated only with RNA 1 plus either the LIYV D RNA M5 or M18, suggesting that these LIYV D RNAs served as templates to generate the P26 subgenomic RNA. The GFP coding region was inserted as an in-frame insertion into the P26 coding region of the LIYV M5 and M18 D RNAs, yielding M5gfp and M18gfp. When transcripts of M5gfp and M18gfp were used to inoculate protoplasts, bright fluorescence was seen only when they were co-inoculated with LIYV RNA 1. The percentage of fluorescent protoplasts ranged from experiment to experiment, but was as high as 5.8%. Time course analyses showed that fluorescence was not detected before 48 h pi, and this correlated with the timing of LIYV RNA 2 and RNA 2 D RNA accumulation, but not with that of LIYV RNA 1
A Leucine Zipper Motif in the Cytoplasmic Domain of gp41 Is Required for HIV-1 Replication and Pathogenesis in Vivo

AbstractA leucine zipper motif is conserved in the cytoplasmic domain of glycoprotein gp41 (gp41c) of all HIV-1 subtypes, but is not present in HIV-2 or SIV. The second leucine residue of the leucine zipper was mutated (L95R) to determine the role of this motif in HIV-1 replication and pathogenesis. The L95R mutant replicated to wild-type levels in activated peripheral blood mononuclear cells and CEMx174 cells. However, L95R replication was impaired in SupT1 cells and in the SCID-hu Thy/Liv mouse. Although the infectivity of wild-type virions and that of L95R mutant virions were equally sensitive to heat treatment, we found that L95R produced more defective virions, due to reduced surface expression and virion incorporation of the env glycoprotein. These results suggest that the L95 residue in the leucine zipper of gp41c of HIV-1 plays an important role in the env expression and virion incorporation that is required for viral replication and pathogenesis in the SCID-hu Thy/Liv mouse. The leucine zipper motif in gp41c may provide a novel anti-HIV-1 target