pinecone/core-2020-05-10-deduplication huggingface dataset:
Internal Cleavage of Hepatitis C Virus NS3 Protein Is Dependent on the Activity of NS34A Protease

AbstractThe nonstructural protein NS3 of the hepatitis C virus (HCV) is indispensable for virus replication and a multifunctional enzyme that contains three catalytic activities such as serine protease, helicase, and NTPase. Here, we demonstrated that the internal cleavage of the HCV NS3 protein occurs in various mammalian cells such as HepG2, COS-7, and NIH3T3. As is observed for the internal cleavage mechanism of the NS3 protein of dengue virus 2, the internal processing of HCV NS3 protein was catalyzed by the active NS3 serine protease and NS4A, but not NS3 alone. From the data acquired from extensive site-directed mutagenesis, we observed that the NS3 protein was internally cleaved at two different sites, FCH1395‖S1396KK and IPT1428‖S1429GD, within RNA helicase domain. The internal cleavage of NS3 protein by NS34A protease was also confirmed in a different isolate of HCV-1b strain. In addition, in vitro transforming assays demonstrated that the internal cleavage product of NS3, NS3a-1, appeared to have higher oncogenic potential than does intact NS3. Taken together, our results suggest that the internal cleavage of NS3 may be associated with the replication and oncogenesis of HCV
Synergistic Interactions of a Potyvirus and a Phloem-Limited Crinivirus in Sweet Potato Plants

AbstractWhen infecting alone, Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) and Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus) cause no or only mild symptoms (slight stunting and purpling), respectively, in the sweet potato (Ipomoea batatas L.). In the SPFMV-resistant cv. Tanzania, SPFMV is also present at extremely low titers, though plants are systemically infected. However, infection with both viruses results in the development of sweet potato virus disease (SPVD) characterized by severe symptoms in leaves and stunting of the plants. Data from this study showed that SPCSV remains confined to phloem and at a similar or slightly lower titer in the SPVD-affected plants, whereas the amounts of SPFMV RNA and CP antigen increase 600-fold. SPFMV was not confined to phloem, and the movement from the inoculated leaf to the upper leaves occurred at a similar rate, regardless of whether or not the plants were infected with SPCSV. Hence, resistance to SPFMV in cv. Tanzania was not based on restricted virus movement, neither did SPCSV significantly enhance the phloem loading or unloading of SPFMV. It is also noteworthy that SPVD is an unusual synergistic interaction in that the potyvirus component is not the cause of synergism but is the beneficiary. It is hypothesized that SPCSV is able to enhance the multiplication of SPFMV in tissues other than where it occurs itself, perhaps by interfering with systemic phloem-dependent signaling required in a resistance mechanism directed against SPFMV
Genetic Dissection of the Multiple Functions of Alfalfa Mosaic Virus Coat Protein in Viral RNA Replication, Encapsidation, and Movement

AbstractCoat protein (CP) of alfalfa mosaic virus (AMV) binds as a dimer to the 3′ termini of the three genomic RNAs and is required for initiation of infection, asymmetric plus-strand RNA accumulation, virion formation, and spread of the virus in plants. A mutational analysis of the multiple functions of AMV CP was made. Mutations that interfered with CP dimer formation in the two-hybrid system had little effect on the initiation of infection or plus-strand RNA accumulation but interfered with virion formation and reduced or abolished cell-to-cell movement of the virus in plants. Six of the 7 basic amino acids in the N-terminal arm of CP (positions 5, 6, 10, 13, 16, and 25) could be deleted or mutated into alanine without affecting any step of the replication cycle except systemic movement in plants. Mutation of Arg-17 interfered with initiation of infection (as previously shown by others) and cell-to-cell movement of the virus but not with plus-strand RNA accumulation or virion formation. The results indicate that in addition to the RNA-binding domain, different domains of AMV CP are involved in initiation of infection, plus-strand RNA accumulation, virion formation, cell-to-cell movement, and systemic spread of the virus
Heparan Sulfate Glycosaminoglycans Are Involved in Adenovirus Type 5 and 2-Host Cell Interactions

AbstractGene therapy vectors derived from subgroup C adenoviruses of the serotype 5 (Ad5) and 2 (Ad2) resulted in inefficient infection of well differentiated respiratory cells, both in vitro and in vivo. The level of expression and localization of the primary receptor for Ad5 and Ad2, termed CAR, do not completely explain why the infection efficiency varies greatly in different experimental conditions. The possibility that additional receptors like proteoglycans are involved in the infection of Ad5 and Ad2 was investigated, because several pathogenic microorganisms use heparan sulfate–glycosaminoglycans (HS-GAGs) as coreceptors for multistep attachment to target cells. The HS-GAG analog heparin decreased Ad5- and Ad2-mediated infection and binding starting from the concentration of 0.1 μg/ml, up to a maximum of 50%. A similar reduction in Ad5 binding and infection was obtained by treatment of cells with heparin lyases I, II, and III but not with chondroitin ABC lyase. The effect of heparin on Ad5 binding has not been observed in surface GAG-defective Raji cells and after treating A549 cells with heparin lyases I, II ,and III. The binding of Ad5 was completely abolished when both CAR was blocked with RmcB antibody and HS-GAGs were competitively inhibited by heparin. Parallel experiments demonstrate that HS-GAGs are irrelevant to binding and infection of the subgroup B adenovirus type 3. Collectively, these results demonstrate for the first time that HS-GAGs expressed on the cell surface are involved in the binding of Ad5 and Ad2 to host cells
Membrane Fusion Activity of Tick-Borne Encephalitis Virus and Recombinant Subviral Particles in a Liposomal Model System

AbstractWe present a kinetic analysis of the membrane fusion activity of tick-borne encephalitis (TBE) virus and TBE-derived recombinant subviral particles (RSPs) in a liposomal model system. Fusion was monitored using a fluorescence assay involving pyrene-labeled phospholipids. Fusion was strictly dependent on low pH, with the optimum being at pH 5.3–5.5 and the threshold at pH 6.8. Fusion did not require a protein or carbohydrate receptor in the target liposomes. Preexposure to low pH of the virus alone resulted in inactivation of its fusion activity. At the optimum pH for fusion and 37°C, the rate and extent of fusion were very high, with more than 50% of the virus fusing within 2 s and the final extent of fusion being 70%. Lowering of the temperature did not result in a significant decrease in the rate and extent of fusion, suggesting that TBE virus fusion is a facile process with a low activation energy, possibly due to the flat orientation of the E glycoprotein on the viral surface facilitating the establishment of direct intermembrane contact. The fusion characteristics of TBE virus and RSPs were similar, indicating that RSPs provide a reliable and convenient model for further study of the membrane fusion properties of TBE virus
The Human Papillomavirus Type 11 E1∧E4 Protein Is Phosphorylated in Genital Epithelium

AbstractThe most abundant viral transcript in human papillomavirus (HPV) 11-infected xenograft tissue has been shown to encode the E1∧E4 protein. The function of E1∧E4 protein has not been determined. Several potential phosphorylation sequence motifs were identified in the HPV 11 E1∧E4 protein, including potential sites of phosphorylation by mitogen-activated protein kinase (MAPK), cAMP-dependent protein kinase (PKA), casein kinase II, and protein kinase C. To test phosphorylation of the HPV 11 E1∧E4 protein, a soluble maltose binding protein (MBP) fusion was produced in Escherichia coli. Only MAPK and PKA phosphorylated the E1∧E4 protein. Phosphoamino acid analysis showed that one or more threonine residues were phosphorylated by MAPK, and both serine and threonine residues were phosphorylated by PKA. MBP–E1∧E4 mutant proteins were designed to delineate the E1∧E4 phosphoacceptor residues. MAPK was shown to phosphorylate E1∧E4 on threonine 53 within a MAPK consensus phorphorylation sequence motif. PKA was shown to phosphorylate E1∧E4 at two residues: threonine 36 within a consensus motif and serine 44 within a variant of the PKA consensus phosphorylation sequence motif. HPV 11-infected human genital tissue grown as a xenograft in an athymic mouse was labeled with [32P]orthophosphate. Phosphoamino acid analysis of E1∧E4 protein immunoprecipitated from 32P-labeled tissue revealed that both serine and threonine residues were phosphorylated. Analysis by liquid chromatography–mass spectrophotometry was consistent with phosphorylation of residues within the PKA and MAPK phosphorylation sequence motifs. Expression of E1∧E4 protein containing phosphorylation substitution mutations showed that the PKA mutant did not differ from wild-type E1∧E4 protein in intracellular distribution. In contrast, the MAPK mutant did not localize exclusively to the cytoplasm nor did it colocalize with wild-type E1∧E4 protein. We conclude that HPV 11 E1∧E4 protein is phosphorylated in vitro and in vivo. Our data are consistent with phosphorylation of HPV 11 E1∧E4 protein by MAPK and PKA in infected tissue
Synthesis of Viral DNA and Late Capsid Protein L1 in Parabasal Spinous Cell Layers of Naturally Occurring Benign Warts Infected with Human Papillomavirus Type 1

AbstractWe investigated human papillomavirus type 1 (HPV1)-specific transcription, viral DNA replication, and viral protein expression in naturally occurring benign tumors by in situ hybridization, 5-bromodeoxyuridine (BrdU) incorporation, and immunohistochemistry and obtained results different from other HPV-infected benign tumors characterized so far. Moderate amounts of transcripts with a putative coding potential for E6/E7, E1, and E2 were demonstrated from the first subrabasal cell layer throughout the stratum spinosum and granulosum. In addition very large amounts of E4 and L1 transcripts were present in the same epithelial layers. This finding was substantiated by the demonstration of L1 and E4 protein already in the bottom-most spinous cell layer. Furthermore massive amplification of the viral DNA as measured by BrdU incorporation and different methods of in situ hybridization took place in the lowest 5 to 10 suprabasal cell layers. These findings are in contrast to the assumption that late gene expression and viral DNA synthesis are restricted to the more differentiated cell layers of the epithelium and point to differences in the regulation of the vegetative life cycle between different papillomavirus types
The E7 Oncoprotein of Human Papillomavirus Type 16 Interacts with F-Actin in Vitro and in Vivo

AbstractWe report here that E7 oncoprotein of human papillomavirus type 16 (HPV-16) forms a complex in vivo and in vitro with actin, one of the components of the cellular cytoskeleton. The in vivo interaction was detected by immunofluorescent staining and confocal microscopic examination of normal human oral keratinocytes (NHOK) and CV-1 cells after transient expression of E7 employing the vaccinia virus–T7 RNA polymerase system and by coimmunoprecipitation from an immortalized, nontumorigenic cell line obtained after transfecting NHOK with the cloned HPV-16 DNA genome. The in vitro interaction was detected by cosedimentation of bacterially expressed E7 phosphorylated with rabbit reticulocyte lysate or purified casein kinase II (CKII) prior to incubation with F-actin. This interaction was inhibited if E7 phosphorylation by the rabbit reticulocyte lysate was prevented with heparin, a CKII inhibitor, or if the amino acids Ser-31 and Ser-32 in E7, which are phosphorylated by CKII, were replaced with amino acids that cannot be phosphorylated. Interestingly, a decrease in the amount of polymerized actin occurred in cells expressing E7
Functional Significance of Alternate Phosphorylation in Sendai Virus P Protein

AbstractPhosphorylation of the negative-sense RNA virus phosphoproteins is highly conserved, implying functional significance. Sendai virus (SV) phosphoprotein (P) is constitutively phosphorylated at S249. Abrogation of the SV P primary phosphorylation causes phosphorylation of P at alternate sites, creating a problem in determining the function of phosphorylation. We have now identified the alternate phosphorylation sites using two-dimensional phosphopeptide analysis of several deletion and point mutants of the P protein. The alternate phosphorylation sites were mutagenized to create P with (S249combo) or without (combo) primary phosphorylation. The combo protein has less than 10% phosphorylation compared with the wild-type P or S249combo. Functional analysis of the mutant proteins using a Sendai virus minigenome replication system showed that the combo P protein was as proficient in supporting minigenome replication as the wild-type P in cell cultures. These studies suggest that like the primary, the alternate phosphorylation of the P protein is also dispensable for virus replication in cell cultures. Interestingly, the ability of the multiple site mutant of P (combo mutant has eight serine residues changed to alanine residues) to support efficient virus RNA synthesis suggests that the P protein has a high flexibility at least in its sequence and perhaps also in structure
Phosphorylation of the RAP74 Subunit of TFIIF Correlates with Tat-Activated Transcription of the HIV-1 Long Terminal Repeat

AbstractTranscription from the HIV-1 long terminal repeat (LTR) is regulated by the viral transactivator Tat, which increases RNA polymerase II (RNAP II) processivity. Previous reports have demonstrated that phosphorylation of the RNAP II carboxy-terminal domain by TFIIH and P-TEFb is important for Tat transactivation. Our present results demonstrate that phosphorylation of the RAP74 subunit of TFIIF is also an important step in Tat transactivation. Interestingly, while the general transcription factor TFIIF is required for both basal and Tat-activated transcription, phosphorylation of the RAP74 subunit occurs in the presence of Tat and correlates with a high level of transcription activity. Using a biotinylated DNA template transcription assay, we provide evidence that RAP74 is phosphorylated by TAFII250 during Tat-activated transcription. Depletion of RAP74 from the HeLa nuclear extract inhibited HIV-1 LTR-driven basal transcription and Tat transactivation. The addition of TFIIF, reconstituted from recombinant RAP30 and RAP74, to the depleted HeLa nuclear extract resulted in restoration of Tat transactivation. Of importance, the exogenous RAP74 was rapidly phosphorylated in the presence of Tat. These results suggest that RAP74 phosphorylation is one important step, of several, in the Tat transactivation cascade
Proline Residues in the HIV-1 NH2-Terminal Capsid Domain: Structure Determinants for Proper Core Assembly and Subsequent Steps of Early Replication

AbstractRecent analyses suggest that the p24 capsid (p24CA) domain of the HIV-1 group-specific antigen (Gag) may be divided into two structurally and functionally distinct moieties: (i) an amino-terminal portion, previously shown to bind the cellular chaperone cyclophilin A, and (ii) a carboxy-terminal domain, known to contribute to the interaction of the Gag and Gag-Pol precursors during the early assembly process. In order to gain deeper insight into the role of the amino-terminal domain of the p24CA protein during viral replication, eight highly conserved proline residues known to promote turns and to terminate α-helices within the p24 tertiary structure were replaced by a leucine residue (P-position-L). Following transfection of the proviral constructs in COS7 cells, the majority of the mutants resembled wild-type viruses with respect to the assembly and release of virions. However, although the released particles contained wild-type levels of genomic viral RNA, the mature products of the Gag and Gag-Pol polyproteins as well as the Env glycoproteins—all of them, except mutant P225L—were either noninfectious or severely affected in their replicative capacity. Entry assays monitoring the process of viral DNA synthesis led to the classification of selected provirus mutants into four different phenotypes: (i) mutant P225L was infectious and allowed complete reverse transcription including formation of 2-LTR circles; (ii) mutants P149L, P170L, and P217L failed to form 2-LTR circles; (iii) mutant P222L displayed a severe defect in binding and incorporating cyclophilin A into virions, was delayed with respect to DNA polymerization, and failed to form a 2-LTR replication intermediate; and (iv) mutant P133L was unable even to synthesize a first-strand cDNA product. All replication-defective mutants were characterized by severe alterations in the stability of virion cores, which were in two cases reflected by visible changes in the core morphology. These results suggest that proline residues in the NH2-terminal capsid domain represent critical structure determinants for proper formation of functional virion cores and subsequent stages of early replication
Role of Matrix Protein in the Type D Retrovirus Replication Cycle: Importance of the Arginine Residue at Position 55

AbstractWe previously reported that a mutant of Mason–Pfizer monkey virus (M-PMV), which has an amino acid substitution in the matrix (MA) protein at position 55, MA-R55W, showed altered viral morphogenesis, reduced glycoprotein incorporation, and loss of infectivity. In this report, we show that two additional amino acid substitutions at this site in MA, R55F and R55Y, also result in similar altered morphogenesis, Env incorporation, and infectivity, demonstrating that these changes are not specific for the substitution of tryptophan in place of arginine 55. Attempts to isolate second site infectious revertants from cells transfected with the R55W mutant genome resulted only in the recovery of infectious viruses in which the codon at position 55 had reverted to one encoding arginine. In contrast, no revertants were obtained from the phenylalanine and tyrosine mutants in which three nucleotide changes had been engineered into the arginine codon. These results confirm that the arginine residue at position 55 is critical for intracellular targeting of M-PMV Gag molecules and support the concept that as part of a cytoplasmic transport retention signal R55 interacts with cellular trafficking components rather than other regions of Gag
Association of Histone Deacetylase with COUP-TF in Tumorigenic Ad12-Transformed Cells and Its Potential Role in Shut-Off of MHC Class I Transcription

AbstractChicken ovalbumin upstream promoter-transcription factor (COUP-TF) is an orphan nuclear receptor that represses transcription of many genes. In adenovirus type 12 (Ad12) transformed cells, a high level of binding activity of COUP-TF to the major histocompatibility complex (MHC) class I enhancer correlates with the down-regulation of class I transcription, which, in turn, contributes to tumorigenesis. The mechanism by which COUP-TF represses transcription has yet to be elucidated. Here we show that COUP-TF represses transcription through its association with histone deacetylase. This was demonstrated using reciprocal binding assays that determined that the interaction between COUP-TF and histone deacetylase requires the COUP-TF C-terminal repression domain. Moreover, a histone deacetylase enzymatic activity was found to be associated with COUP-TF in Ad12-transformed cells. Transfection experiments further revealed that exogenous histone deacetylase facilitates transcriptional repression by COUP-TF. Also, supershift assays suggest that the transcriptional corepressor N-CoR, which is known to associate with histone deacetylases, is a part of the COUP-TF complex bound to the MHC class I enhancer R2 site. Finally, we provide evidence that inhibition of histone deacetylases relieves the repression of MHC class I expression in Ad12-transformed cells. Taken together these results support the notion that deacetylation of histones, mediated through COUP-TF, serves to down-regulate MHC class I transcription in Ad12-transformed cells
Involvement of the Aphthovirus RNA Region Located between the Two Functional AUGs in Start Codon Selection

AbstractInitiation of translation in picornavirus RNAs occurs internally, mediated by an element termed internal ribosome entry site (IRES). In the aphthovirus RNA, the IRES element directs translation initiation at two in-frame AUGs separated by 84 nucleotides. We have found that bicistronic constructs that contained the IRES element followed by the fragment including the aphthovirus start codons in front of the second gene mimicked the translation initiation pattern of viral RNA observed in infected cells. In those constructs, the frequency of initiation at the first AUG was increased by a sequence context that resembled the favorable consensus for cap-dependent translation, although initiation at the second site was always preferred. In addition, we have found that initiation at the second start codon was not diminished under conditions in which the first initiation codon was blocked by antisense oligonucleotide interference. Interestingly, mutations that positioned the second AUG out-of-frame with the first AUG did not interfere with the frequency of initiation at the second one. On the contrary, IRES-dependent translation initiation in bicistronic constructs lacking the sequences present between functional AUGs in the viral RNA was sensitive to the presence of out-of-frame initiator codons and hairpins in the spacer region. This remarkable difference in start codon recognition was due to the nucleotide composition of the RNA that separated the IRES from the initiator codon. Thus our results indicate that the region located in the aphthovirus RNA between functional AUGs is involved in start codon recognition, strongly favoring selection of the second start AUG as the main initiator codon
U937 Cells Overexpressing bcl-xl Are Resistant to Human Immunodeficiency Virus-1-Induced Apoptosis and Human Immunodeficiency Virus-1 Replication

AbstractMany viruses, including human immunodeficiency virus type 1 (HIV-1), induce apoptosis and are affected by cellular expression of antiapoptotic genes. We sought to examine the effect of antiapoptotic gene expression on HIV replication by transfecting the promyelomonocytic cell line U937 with the bcl-xl gene to obtain clones of U937 cells that overexpressed bcl-xl (designated U937bcl-xl), a negative control U937 clone transfected with vector alone (designated U937neo) and a clone overexpressing bcl-2 (designated U937bcl-2). After infection with HIV-1, U937neo cells underwent apoptosis four times as frequently as the U937bcl-xl cells. Furthermore, U937bcl-xl cells produced 5-fold less HIV-1 protein than U937neo, whereas U937bcl-2 produced at least 2-fold more p24 than the U937neo control. Transient coexpression of bcl-2 or bcl-xl decreased HIV production and transcription from the HIV LTR. To define the mechanism by which bcl-xl, but not bcl-2, inhibits HIV expression, we examined bcl-2 and bcl-xl expression after HIV infection and CD4 cross-linking. Although HIV-1 infection or cross-linking CD4 led to a decrease in expression of bcl-2, it had no effect on bcl-xl expression. These results provide a mechanism for the resistance of U937bcl-xl transfectants, but not U937bcl-2 transfectants, to HIV-1 replication in monocytic cellsin vitro. Therapies that up-regulate bcl-xl expression potentially provide a novel means to decrease the destructiveness of HIV-1