pinecone/core-2020-05-10-deduplication huggingface dataset:
Analysis, Quantification, and Evolutionary Consequences of HIV-1 in Vitro Recombination

AbstractHIV-1 recombination was studied in vitro by viral cocultivation of four combinations of strains of subtypes B, D, and F. Viral cocultivations were performed in MT-4 cells and maintained for 22 days. The parental and recombinant forms were quantified by a specific PCR system in an env fragment of 2500 nucleotides. On day 5, there was a close correlation between the proportion of recombination and the genetic distance between strains. In three of the four viral combinations studied, a steady increase in the proportion of recombinant genomes was observed over time. This rise coincided with the progressive loss of one of the parental strains, resulting in less diverse viral populations. Nucleotide sequencing of biological recombinant clones from the B/D cocultivation revealed a higher number of recombination events in pol than in env gene, and an increasing number of crossovers per clone with time
Polymorphism in the Infectious Salmon Anemia Virus Hemagglutinin Gene: Importance and Possible Implications for Evolution and Ecology of Infectious Salmon Anemia Disease

AbstractInfectious salmon anemia (ISA) is an emerging disease in farmed Atlantic salmon with important commercial consequences. The pathogenicity of the ISA virus (ISAV; an orthomyxovirus) varies, observed as differences in disease development and clinical signs. A small polymorphic region (PR) in the ISAV genomic segment encoding the hemagglutinin (HA) has been described. An analysis of 33 HA gene sequences from historical and recent ISA outbreaks was performed, added to a selection of previously published HA sequences. A differential deletion model explaining the generation of HA polymorphism is proposed. The European ISAV sequences could be grouped according to deletion patterns in PR. Cell-culture replication and cytopathic effect varied between viruses from different PR groups. A rather complex epidemiology is suggested, as (a) HA sequences representing several PR variants were detected in three samples; (b) identical mutations occurred in different genetic lineages; and (c) large genetic differences were present in closely related viruses
Effects of 1,8-Diaminooctane on the FIV Rev Regulatory System

AbstractProper function of the Rev regulatory system is essential for the replication of lentiviruses, including feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV-1). Specifically, Rev affects the overall stability of viral mRNAs that encode necessary structural and enzymatic proteins. In turn, the eukaryotic initiation factor (eIF-5A) is indispensable for Rev function and is the only known protein whose biologically active form requires the unique amino acid, hypusine. Because 1,8-diaminooctane blocks the formation of hypusine by disrupting the cellular enzyme, deoxyhypusine synthase, thereby preventing activation of eIF-5A, we investigated the effects of 1,8-diaminooctane on posttranscriptional regulation. These are the first results to demonstrate that diaminooctane significantly reduced viral replication in a dose-dependent manner, even under conditions of contact inhibition, diminishing the compound's effect on cell proliferation. Similarly, the addition of increased concentrations of diaminooctane caused a reduction in the expression of a Rev-dependent CAT system without affecting a Rev-independent CAT system. At the RNA level, exposure of chronically infected CrFK cells to increasing concentrations of diaminooctane substantially decreased the levels of unspliced and singly spliced viral mRNAs and increased the relative amounts of multiply spliced transcripts in the cytoplasm. The findings of this study are the first demonstration that FIV, similar to HIV-1, requires eIF-5A for efficient Rev function and that small molecule intervention can indirectly target this lentivirus regulatory system
Rabbit Hemorrhagic Disease Virus: Identification of a Cleavage Site in the Viral Polyprotein That Is Not Processed by the Known Calicivirus Protease

AbstractThe positive-strand RNA genome of the calicivirus rabbit hemorrhagic disease virus (RHDV) contains one long open reading frame (ORF) covering almost 95% of the genomic RNA. Translation of this ORF leads to a polyprotein that is proteolytically processed at eight sites. Site nos. 4 and 5 from the amino-terminus are located within the protein p41 and obviously belong to alternative cleavage pathways leading to p23/2 and p18 or p29 and p13. Seven of the eight cleavage sites were identified before and the flanking sequences fulfill the requirements of the known RHDV protease, so that it is very likely that all theses sites are cleaved by this enzyme. The last unknown cleavage site was no. 4, one of the two alternative sites within p41 that separates the nonstructural proteins p23/2 and the VPg precursor p18. Mutagenesis studies identified aspartic acid at position 936 and arginine at position 937 as the residues that flank the cleavage site. The sequence at the processing site is unusual for the RHDV 3C-like protease since other sites display glutamic acid or glutamine at the P1 site and glycine, aspartic acid, or threonine at the P1′ site. Expression of a polyprotein fragment lacking the viral protease revealed that the newly identified site is not cleaved by the RHDV protease but by an unknown proteolytic activity
Transmission of Human Cytomegalovirus from Infected Uterine Microvascular Endothelial Cells to Differentiating/Invasive Placental Cytotrophoblasts

AbstractAnalysis of placentas infected with human cytomegalovirus (CMV) suggested that viral transmission could involve differentiating/invasive cytotrophoblasts in villi that attach the placenta to the uterine wall. To parse the cellular components in this process, we developed a coculture system of polarized uterine microvascular endothelial cell (UtMVEC) infection with an endothelial cell–tropic pathogenic strain of CMV. Then we evaluated the potential role of neutrophils and endothelial cells in the spread of infection to differentiating cytotrophoblasts. As shown by immunocytochemistry and analysis of viral replication, CMV preferentially infected endothelial cells via apical membranes and disrupted cell junction proteins, thereby altering paracellular permeability and cell polarity. Neutralizing antibodies to CMV glycoprotein B, an envelope component that facilitates virion penetration, blocked plaque formation in polarized UtMVEC. Neutrophils transmitted CMV infection to UtMVEC, which in turn infected cytotrophoblasts. However, neutrophils did not directly infect cytotrophoblasts. These findings implicate endothelial cells from the uterine microvasculature as a potential source for CMV infection of endovascular cytotrophoblasts of the anchoring villi. Possibly the cytokine/chemokine milieu in the pregnant uterus could attract immune cells that infect endothelial cells in hybrid fetal–maternal vessels. In turn, these cells could infect endovascular cytotrophoblasts, one possible initiation point of a cascade that results in retrograde placental CMV infection
Replication of Potato spindle tuber viroid in Cultured Cells of Tobacco and Nicotiana benthamiana: The Role of Specific Nucleotides in Determining Replication Levels for Host Adaptation

AbstractWe have developed an electroporation protocol to inoculate cultured cells of tobacco and Nicotiana benthamiana with in vitro transcripts of Potato spindle tuber viroid (PSTVd) to characterize viroid structural features that determine replication efficiency at the cellular level. Both (+)- and (−)-strands of PSTVd were detected by Northern blots as early as 6 h postinoculation (h.p.i.). Accumulation of the (+)-circular PSTVd increased very rapidly starting at 24 h.p.i. and continued beyond 6 days postinoculation. Viroid accumulation in individual cells was visualized by in situ hybridization, which showed that 60–70% of the cells were infected. Previous work showed that C259 → U substitution converted tomato isolate PSTVdKF440-2 into a strain that is infectious on tobacco (M. Wassenegger, R. L. Spieker, S. Thalmeir, F.-U. Gast, L. Riedel, and H. L. Sänger, 1996. Virology 226, 191–197). Similarly, C259 → U or U257 → A substitution in the Intermediate strain (PSTVdInt) conferred infectivity in tobacco (Y. Zhu, Y. Qi, Y. Xun, R. Owens, and B. Ding, 2002. Plant Physiol. 130, 138–146). Our replication assays in tobacco-cultured cells demonstrated that U257 → A and C259 → U substitutions each enhanced PSTVd replication by 5- to 10-fold. Replacement of U257 with C, but not with G, also led to enhanced replication in tobacco cells. Replacement of C259 with nucleotide A or G did not enhance replication. Elevated accumulation of the (−)- and (+)-strands of these mutants was in part due to enhanced transcription. Interestingly, all of the nucleotide changes did not alter PSTVd replication levels in N. benthamiana cells. These results provide insights about PSTVd structures that modulate replication efficiency in adapting to a specific host
Structural Basis for Ubiquitin-like ISG 15 Protein Binding to the NS1 Protein of Influenza B Virus: A Protein–Protein Interaction Function That Is Not Shared by the Corresponding N-terminal Domain of the NS1 Protein of Influenza A Virus

AbstractThe N-terminal domains of the NS1 protein of influenza B virus (NS1B protein) and the NS1 protein of influenza A virus (NS1A protein) share one function: binding double-stranded RNA (dsRNA). Here we show that the N-terminal domain of the NS1B protein possesses an additional function that is not shared by its NS1A counterpart: binding the ubiquitin-like ISG15 protein that is induced by influenza B virus infection. Homology modeling predicts that the dimeric six-helical N-terminal domain of the NS1B protein differs from its NS1A protein counterpart in containing large loops between helices 1 and 2 (loops 1 and 1′) and between helices 2 and 3 (loops 2 and 2′). Mutagenesis establishes that residues located in loop 1/1′ together with residues located in polypeptide segment 94–103 form the ISG15 protein-binding site of NS1B protein. Loop 1/1′ is not required for dsRNA binding, which instead requires arginine residues R50, R53, R50′, and R53′ located in antiparallel helices 1 and 1′. Further, we demonstrate that the binding sites for RNA and protein are independent of each other. In particular, ISG15 and dsRNA can bind simultaneously; the binding of the ISG15 protein does not have a detectable effect on the binding of dsRNA, and vice versa
Persistence of Simian Varicella Virus DNA in CD4+ and CD8+ Blood Mononuclear Cells for Years after Intratracheal Inoculation of African Green Monkeys

AbstractSimian varicella virus (SVV) DNA was detected in blood mononuclear cells (MNCs) of adult African green monkeys 7 days to 23 months after intratracheal inoculation with 103 plaque forming units. Infectious virus was not detected in MNCs at 14 months postinfection (p.i.), and electron microscopic (EM) analysis of MNCs from two monkeys 21 months p.i. did not reveal virus particles. Real-time quantitative PCR analysis of DNA from blood MNCs taken at multiple intervals from SVV-infected monkeys M7 and M8 revealed a 10- to 100-fold decrease, but not clearance of SVV DNA in MNCs between 11 and 17 months p.i. Thereafter, the SVV DNA copy number did not decrease further between 17 and 23 months p.i. PCR analysis of MNCs sorted by flow cytometry revealed SVV DNA in T cells (CD4+, CD8+) and B cells (CD20+), but not in monocyte-macrophages (CD14+), 10 days p.i. At 11 and 23 months p.i., SVV DNA was found exclusively in CD4+ and CD8+ T cells. Whether the detection of SVV DNA in CD4+ and CD8+ MNCs many months after the resolution of acute varicella reflects continued infection of these cells that began at the time of acute varicella or represents infection acquired by MNCs trafficking through infected tissues is unknown
Cell-Culture Propagation of Porcine Enteric Calicivirus Mediated by Intestinal Contents Is Dependent on the Cyclic AMP Signaling Pathway

AbstractEnteric caliciviruses are emerging pathogens in humans and animals, but they do not replicate in cell culture except for the porcine enteric calicivirus (PEC) Cowden strain. The PEC Cowden strain grows in pig kidney (LLC-PK) cells, but only in the presence of intestinal contents (IC) from uninfected gnotobiotic pigs in the medium. In this study, we investigated the relationship between IC and growth of Cowden PEC. Pretreatment of cells or the virus with IC or transfection of viral RNA into cells did not induce virus growth unless the medium was supplemented with IC. Among modulators of cell signal transduction, the G protein uncoupler, suramin, adenylate cyclase (AC) inhibitor, MDL-12,330A, and the cAMP-dependent protein kinase (PKA) inhibitor, N-(2-[bromocinnamulamino]ethyl)-5-isoquinolinesulfonamide (NBEI) inhibited the effect of IC on virus growth for up to 72 h. These data indicate that PEC virus replication may be dependent on an initial cAMP signaling pathway induced by IC
Growth Arrest of HPV-Positive Cells after Histone Deacetylase Inhibition Is Independent of E6/E7 Oncogene Expression

AbstractInhibitors of histone deacetylase (HDAC) are capable of arresting growth in cervical carcinoma cells in the G1 phase of the cell cycle. Although HPV E6/E7 mRNA steady-state levels appeared to be constant after prolonged treatment, time-course experiments revealed that viral transcription was transiently down-regulated between 7–10 h prior to cdk2 suppression. To test whether transitory suppression was a prerequisite for the biological outcome after HDAC inhibition, we took advantage of two immortalized human keratinocyte cell lines in which E6/E7 oncogene expression was controlled by different regulatory regions. After treatment with sodium butyrate (NaB) or trichostatin A (TSA), HPV16 upstream regulatory region (URR)-directed transcription was down-regulated, showing kinetics similar to those in cervical carcinoma cells. In contrast, β-actin promoter controlled E6/E7 transcription was even temporarily increased and finally declined to levels initially detected in the untreated controls. Both cell lines, however, were arrested in G1 and showed complete suppression of cdk2 activity that was preceded by a strong up-regulation of the cdk2 inhibitors p21CIP1 and p27KIP1. These results demonstrate that growth of HPV16/18-positive cells can be arrested by HDAC inhibitors despite ongoing HPV transcription and thus independently of any potential position effects uncoupling URR-directed gene expression by adjacent cellular promoters or by downstream 3′-polyadenylation sites after viral integration into the host genome during multistep carcinogenesis
Characterization of the Oligomerization Domain of the Phosphoprotein of Human Parainfluenza Virus Type 3

AbstractThe phosphoprotein (P) of human parainfluenza virus type 3 (HPIV 3) plays a central role in the viral genome RNA transcription and replication. It acts as an essential cofactor of the RNA polymerase (L) by forming a functional L-P complex, binds to the genomic N-RNA template to recruit the L-P complex for RNA synthesis, and interacts with the nucleocapsid protein (N) to form the encapsidation complex (N-P). We have earlier demonstrated that the P protein forms oligomers (B. P. De, M. A. Hoffman, S. Choudhary, C. C. Huntley, and A. K. Banerjee, 2000, J. Virol. 74, 5886–5895) and in this article we identified the putative oligomerization domain of the P protein and studied the role of this domain in transcription. By computer analyses, we have localized a high-score coiled-coil motif characteristic of oligomerization domain residing between the amino acid residues 423 and 457 of the P protein. Deletion of 12 amino acid residues within this coiled-coil motif (PΔ439–450) completely abrogated oligomerization, whereas deletion in other regions outside the motif had no significant effect. The mutant PΔ439–450 was both defective in mRNA synthesis in vitro and minigenome transcription in vivo. Interestingly, the mutant interacted with L to form L-P complex, albeit less efficiently, while its interaction with N protein to form N-P complex and with N-RNA template was similar to the wt P protein. Our results indicate that oligomerization provides a key function to the P protein in the transcription of HPIV 3 genome RNA
Association of Influenza Virus Matrix Protein with Ribonucleoproteins May Control Viral Growth and Morphology

AbstractThe matrix protein (M1) of influenza virus plays a central role in viral replication. In relation to viral growth and morphology, we studied the RNP-binding activity of M1s from high-growth strain A/Puerto Rico/8/34 (A/PR8/34) and the relatively low-growth wild-type strain A/Nanchang/933/95. The RNP-binding strength of M1 was studied by disruption of M1 from M1/RNP complexes with salt and acidic condition. Our results indicated that binding of M1 of high-growth A/PR8/34 was more difficult to break than the binding of M1 of low-growth A/Nanchang/933/95. Consistent with the presence of M1 in A/PR8/34, binding of M1 of Resvir-9, a reassortant containing P, M, and NS genes from A/PR8/34 and the rest of genes from A/Nanchang/933/95 and retaining relative high-growth characteristic, was relatively difficult to break than the binding of M1 of A/Nanchang/933/95. Physical properties of morphological features of these viruses were studied by velocity sucrose gradient centrifugation and transmission electron microscopy of purified viral particles, and by immunofluorescence staining of hemagglutinin expressed on the surface of infected cells. The results demonstrated that high-growth strains, A/PR8/34, and a relative high-growth reassortant, Resvir-9, had characteristics associated predominantly with spherical particles, while the low-growth strain, A/Nanchang/933/95, had characteristics of filamentous particles. These studies indicate that the binding between M1 and RNP complex might determine viral growth and morphology
Interaction of Human Papillomavirus Type 16 L2 with Cellular Proteins: Identification of Novel Nuclear Body-Associated Proteins

AbstractTwo structural proteins form the Papillomavirus (PV) capsids. While the functions of the major structural protein L1 are well established, the exact functions for the minor structural protein L2 are much less well defined, except for some information on a role in viral entry and maturation of infectious virions. To gain more insight in the function of L2 we used the yeast two hybrid system with the Human Papillomavirus (HPV) 11 L2 and HPV16 L2 as bait proteins to isolate putative cellular interaction partners. We identified four proteins interacting with L2 proteins of at least two different HPV types and this interaction was confirmed in vitro by pull-down assays. Further evidence for this interaction was obtained by in vivo localization studies. Two of the proteins, the previously described PATZ and a novel protein, designated PLINP, were localized in discrete nuclear domains and colocalized with L2. The third protein, designated PMSP, is a newly identified cytoplasmic protein which was recruited to nuclear dots when coexpressed with L2. The fourth protein interacting with HPV16, 11 and 1 L2, the tubular-nephritis antigen related protein (TIN-Ag-RP), shows a cytoplasmic as well as a membrane bound subcellular distribution. Taken together, our data indicate that L2 of HPVs with different phenotypes interacts with several cellular host proteins, recruits one of them to the nucleus, and is complexed with at least three cellular proteins in specific nuclear domains. These findings suggest an HPV type-independent modulatory function of L2 on host-cell functions that involves discrete nuclear domains and alteration of the subcellular distribution of cellular proteins. The interacting cellular proteins identified may play a role in the viral life cycle and establishment of viral persistence
High-Affinity Rep-Binding Is not Required for the Replication of a Geminivirus DNA and Its Satellite

AbstractThe 682-nt satellite DNA (sat-DNA) of Tomato leaf curl virus (TLCV) depends on the helper virus for its replication. In contrast to the strict specificity that exists in each geminivirus for its cognate replication associated protein (Rep), TLCV sat-DNA can utilize Rep encoded by distinct geminiviruses. We have used a combination of protein-binding assays and mutagenesis to show that repeat motifs in TLCV and sat-DNA are essential for Rep-binding in vitro. Surprisingly, mutants of TLCV and sat-DNA impaired in their ability to bind TLCV Rep in vitro were infectious in tomato. Thus, in contrast to other geminiviruses reported, TLCV and sat-DNA replication is independent of the high-affinity in vitro Rep binding. These results prompt a reassessment of the current model of geminivirus replication where Rep/DNA interaction is a highly specific step in the initiation of rolling circle replication
A New Parvovirus Genotype Persistent in Human Skin

AbstractParvovirus B19 is the exclusive human pathogen of the Erythrovirus genus. In classical view, the B19 DNA sequence shows little variability, with no disease-specific or tissue type specific associations. We examined skin biopsies from patients with B19-unrelated skin disease or from constitutionally healthy adults by polymerase chain reaction assays for four different genomic regions of the B19 virus. Sequencing showed that the skin-derived viral DNA differed within the protein-coding region from the B19 reference sequences by 10.8% and from the V9 variant by 8.6% and within the noncoding region (covering nucleotides 189–435 of the promoter region) by 26.5 and 17.2%, respectively. Despite this sequence difference, the promoter region was shown by a luciferase gene expression assay to be biologically active. We have detected a new B19 virus genotype, K71, which differs extensively from the known B19-virus genotypes and is persistently carried in human skin
The Canine Minute Virus (Minute Virus of Canines) Is a Distinct Parvovirus That Is Most Similar to Bovine Parvovirus

AbstractWe characterized the genome and proteins of the canine minute virus (the minute virus of canines (MVC)). The genome sequence showed MVC to be an autonomous parvovirus encoding a large nonstructural protein 1 gene, a smaller nonstructural protein, and overlapping VP1 and VP2 protein genes. The virus was most closely related to bovine parvovirus (BPV), with which it was 43% identical at the DNA sequence level, while the NS1 and VP1 proteins were 33.6 and 41.4% identical to those of BPV, respectively. Spliced messages of the NS1 gene transcripts were detected by RT-PCR. VP1 and VP2 proteins were detected in purified capsids, as were modified versions of each protein, and VP3 was also found in full capsids