pinecone/core-2020-05-10-deduplication huggingface dataset:
A Novel Link between Stress and Human Cytomegalovirus (HCMV) Infection: Sympathetic Hyperactivity Stimulates HCMV Activation

AbstractRecently, inflammatory mediators such as TNFα were identified as triggering active human cytomegalovirus (HCMV) infection. Here, we demonstrate that a highly stressful event in the absence of systemic inflammation, as observed in patients with acute myocardial infarction, leads to the development of an active HCMV infection in latently infected patients. Elucidating the molecular mechanism of virus activation, we could show that catecholamines directly stimulate the HCMV immediate-early (IE) enhancer/promoter in monocytic cells via β-2 adrenergic receptors. Subsequent activation of the cAMP/PK-A-signaling pathway results in enhanced synthesis and binding of the transcription factor CREB-1/ATF-1 to the cAMP-responsive elements within the IE enhancer. Epinephrine also enhanced HCMV gene expression in infected THP-1 cells by about 50% in three of four experiments. These data suggest that HCMV, like HSV-1 and VZV, can be (re)activated under stress conditions
An Anti-Human Immunodeficiency Virus Multiple Antigen Peptide Encompassing the Cleavage Region of the Env Precursor Interferes With Membrane Fusion at a Post-CD4 Binding Step

AbstractCLIV is a multiple antigen peptide ([PTKAKRRVVQREKR]4-K2-K-βA) that encompasses the cleavage region of the human immunodeficiency virus type 1 (HIV-1) envelope precursor. It displays an antiviral activity against HIV-1 and HIV-2 and inhibits HIV-1 Env-mediated cell-to-cell fusion. This effect has previously been attributed to interference with Env processing, resulting in the expression of a nonfusogenic envelope [Virology (1998) 247, 137]. However, we show here that CLIV does not alter the status of Env cleavage at steady state. Using various aggregation/syncytium assays that allow us to discriminate between gp120/CD4 binding and binding followed by gp41-mediated fusion, we demonstrate that CLIV inhibits a step of the cell-to-cell fusion process after CD4 binding. We demonstrate also that CLIV binds at 37°C to a single class of protein present at the CD4+ cell surface (Scatchard analysis: Kd = 8 nM; Bmax = 104 sites/cell) and that the fusion inhibition activity seems to correlate with binding to this proteic component. In contrast, CLIV interacts with neither membrane-inserted nor CD4-associated Env. We therefore propose that CLIV interferes after Env/CD4 binding with a step of the membrane fusion process that may involve the C-terminal domain of gp120
Analysis of RNA Secondary Structure in Replication of Human Parainfluenza Virus Type 3

AbstractThe terminal RNA regions of the genomic and antigenomic RNAs of the paramyxoviruses and rhabdoviruses are known to contain sequences essential for RNA replication and transcription. The 3′- and 5′-termini of human parainfluenza virus type 3 (HPIV3) genomic RNA, termed leader and trailer sequences, respectively, are capable of forming stable stem-loop structures. Additionally, the 17 terminal bases of the leader and trailer are complementary and therefore also capable of forming a helical structure. We investigated the roles of the stem-loop structure and terminal complementarity in HPIV3 RNA replication and transcription in vivo using a minigenome containing all RNA elements necessary for these processes. By mutational analysis, we show that the RNA secondary structure features present at the termini of HPIV3 have no discernible role in replication or transcription. Rather, the primary sequence of these regions is what is critical in promoting replication. Interestingly, a mutation at leader base 24 was found to revert a mutation at leader position 5 but probably not via RNA secondary structure restoration
In Vitro Processing of Human Immunodeficiency Virus Type 1 Gag Virus-like Particles

AbstractHuman immunodeficiency virus (HIV) Gag proteins are assembled into virus particles and then cleaved by the virion-associated HIV protease. Concomitant with Gag processing, doughnut-like HIV particles (the immature form) are converted to particles containing condensed cores (the mature form). Here we describe the in vitro processing of immature HIV Gag virus-like particles (VLP) by exogenously added HIV protease. Following delipidization, sequential processing of immature VLP showed that the matrix (MA)/capsid (CA) junction was cleaved faster than the CA/nucleocapsid (NC) junction, an altered order of processing when compared with authentic processing. When the in vitro processed VLP were analyzed on density gradients, most of the MA, CA-p15 intermediate, and NC were detected as a highly multimeric form, equivalent to the unprocessed VLP. In contrast, CA was found as a monomer dissociated from the multimeric CA-p15 following cleavage of the CA/NC junction. Electron microscopy revealed that the in vitro processing was accompanied by conversion of the doughnut-like particles to particles containing condensed cores and spherical outer shells. The cores, however, lacked core shells, which are normally observed for authentic HIV, suggesting that the in vitro processing of immature VLP failed to produce core shells
Characterization of Low Virulent Strains of Highly Pathogenic A/Hong Kong/156/97 (H5N1) Virus in Mice after Passage in Embryonated Hens' Eggs

AbstractAvian influenza A H5N1 viruses were isolated from humans for the first time in Hong Kong in 1997. The virulence of A/Hong Kong/156/97 (HK156) strain in mice was found to change significantly depending on the passage history of the virus. Madin-Darby canine kidney (MDCK) cell-grown parental virus and three of its clones derived from mouse brain showed high pathogenicity in mice after intranasal or intracerebral infection. In contrast, the egg-derived parental virus HK156-E3 and its cloned viruses were markedly less pathogenic in mice. It appeared that differences in pathogenicity among viruses derived from MDCK cells and eggs were due to their ability or inability to disseminate from the lungs to the brain. Sequence analysis of the entire protein coding regions of all eight RNA genome segments revealed a total of six conserved amino acid differences in the HA1 domain (residue 211) of the HA protein, as well as the PB1 (residues 456 and 712), PA (residue 631), NP (residue 127), and NS1 (residue 101) proteins that correlated with observed changes in virulence and neurovirulence of HK156 virus in mice. Thus it was evident that the passaging of HK156 in embryonated eggs led to the adaptation and selection of variants demonstrating markedly decreased pathogenicity and neurovirulence in mice that appeared to be attributable to specific amino acid changes in the HA and internal proteins
Long Nucleotide Insertions between the HN and L Protein Coding Regions of Human Parainfluenza Virus Type 3 Yield Viruses With Temperature-Sensitive and Attenuation Phenotypes

AbstractRecombinant parainfluenza virus 3 (rPIV3) is being developed as a vector to express foreign genes as a bivalent or multivalent live attenuated virus vaccine. In the present study, we examined the effect of inserted foreign sequence on virus replication in vitro and in vivo, focusing on the parameter of insert length. In one type of construct, foreign sequence of increasing length was flanked by PIV3 transcription signals and inserted as an additional gene unit (GU insert) between the HN and L genes, so that one additional mRNA would be made. In a second type of construct, foreign sequence was inserted into the downstream NCR (NCR insert) of the HN gene, so that the number of encoded mRNAs remained unchanged. In each case, the foreign sequence was designed to lack any significant open reading frame, which permitted an evaluation of the effect of insert length on replication independent of an effect of an expressed protein. The GU or NCR insert sizes ranged from 168 nucleotides (nt) to 3918 nt. rPIV3s containing GU insertions of up to 3918 nt in length, the largest size tested, were viable and replicated efficiently at permissive temperatures in vitro, but a reduction in plaque size was seen at 39°C and 40°C. The rPIV3 with a 3918-nt GU insertion was restricted in replication in the upper (fivefold) and lower (25-fold) respiratory tracts of hamsters. Although a 1908-nt GU insertion did not significantly modify replication of wild-type PIV3 in vitro or in vivo, its introduction significantly augmented the level of temperature sensitivity (ts) and attenuation (att) specified by three mutations in the L protein of a cold-passaged attenuated PIV3 vaccine virus. rPIV3s bearing a 3126- or 3894-nt NCR insertion exhibited in vitro and in vivo phenotypes like those of the rPIV3s bearing similar-sized GU insertions. These findings indicate that rPIV3s whose genome length has been increased by more than 3000 nt by either a GU or an NCR insertion exhibit an unexpected host-range phenotype, that is, efficient replication in vitro but restricted replication in hamsters, especially in the lower respiratory tract. Furthermore, these effects were greatly enhanced when the rPIV3 backbone contained other ts or att mutations. The implications of these findings for the use of single-stranded, negative-sense RNA viruses as vectors for vaccines are discussed
The Three-Dimensional Structure of Bacteriophage PP7 from Pseudomonas aeruginosa at 3.7-Å Resolution

AbstractThe three-dimensional structure of phage PP7 from Pseudomonas aeruginosa has been determined to 3.7-Å resolution. A comparison with distantly related small RNA phages showed that the biggest differences were found in the FG loops, forming the contacts around the fivefold and threefold axes. In contrast to the situation in other phages, the FG loops of phage PP7 are very similar in all three subunits. This supports the hypothesis that no switches are needed for the assembly control in these viruses. Some of the most conserved residues lie within the region involved in RNA binding in the related phages MS2 and seem to have the same function
Characterization of Φ8, a Bacteriophage Containing Three Double-Stranded RNA Genomic Segments and Distantly Related to Φ6

AbstractThe three double-stranded RNA genomic segments of bacteriophage Φ8 were copied as cDNA, and their nucleotide sequences were determined. Although the organization of the genome is similar to that of Φ6, there is no similarity in either the nucleotide sequences or the amino acid sequences, with the exception of the motifs characteristic of viral RNA polymerases that are found in the presumptive polymerase sequence. Several features of the viral proteins differ markedly from those of Φ6. Although both phages are covered by a lipid-containing membrane, the protein compositions are very different. The most striking difference is that protein P8, which constitutes a shell around the procapsid in Φ6, is part of the membrane in Φ8. The host attachment protein consists of two peptides rather than one and the phage attaches directly to the lipopolysaccharide of the host rather than to a type IV pilus. The host range of Φ8 includes rough strains of Salmonella typhimurium and of pseudomonad
SNDV, a Novel Virus of the Extremely Thermophilic and Acidophilic Archaeon Sulfolobus

AbstractWe describe a novel virus, SNDV (Sulfolobus neozealandicus droplet-shaped virus), of the crenarchaeotal archaeon Sulfolobus, which was found in a carrier state in a Sulfolobus strain isolated from a field sample from New Zealand. SNDV particles are droplet-shaped and densely covered by thin tail fibers at their pointed ends. The virion consists of a core and a coat. The latter has the appearance of a beehive and has a surface that is either helically ribbed or a stack of hoops. The genome is cccDNA of 20 kb, which is modified by dam-like methylation. It is cleaved by only a few type II restriction enzymes e.g., DpnI but not MboI, demonstrating an N(6)-methylation of the adenine residue in GATC sequences. The DNA-modifying system differentiates between virus and host. We postulate a virus-encoded methylase that is active on hemimethylated DNA. The host range of SNDV is confined to few Sulfolobus strains from New Zealand. The virus persists in an unstable carrier state rather than as a prophage. Due to its uniqueness we propose to assign it to a novel virus family termed Guttaviridae
The Cowpox Virus Serpin SPI-3 Complexes with and Inhibits Urokinase-Type and Tissue-Type Plasminogen Activators and Plasmin

AbstractThe orthopoxvirus serpin SPI-3 is N-glycosylated and suppresses fusion between infected cells. Although SPI-3 contains motifs conserved in inhibitory serpins, no proteinase inhibition by SPI-3 has been demonstrated, and mutations within the serpin reactive center loop (RCL) do not affect the ability to regulate cell fusion. We demonstrate here that SPI-3 protein expressed by transcription/translation in vitro is able to form SDS-stable complexes with the serine proteinases plasmin, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA), consistent with inhibitory activity of the serpin. Weaker complexes were noted with factor Xa and thrombin. Mutation of Arg-340/Ser-341 at the predicted P1/P1′ sites within the RCL prevented the formation of complexes between SPI-3 and plasmin, uPA, or tPA, suggesting that the arginine at the P1 position was required for complex formation. SPI-3 protein lacking the N-terminal signal peptide was purified by means of an N-terminal His10-tag and gave complete inhibition in vitro of plasmin, uPA, and tPA and partial inhibition of factor Xa. SPI-3 is therefore a bifunctional protein that acts as a proteinase inhibitor and suppresses infected cell–cell fusion. As a proteinase inhibitor, SPI-3 has similar specificity to the leporipoxvirus SERP1 protein of myxoma virus, although the two serpins are less than 30% identical overall. The inhibition constants of SPI-3 for plasmin, uPA, and tPA were determined to be 0.64, 0.51, and 1.9 nM, respectively, very similar to the corresponding Ki values of SERP1
The Cowpox Virus SPI-3 and Myxoma Virus SERP1 Serpins Are Not Functionally Interchangeable despite Their Similar Proteinase Inhibition Profiles in Vitro

AbstractThe myxoma virus (MYX) serpin SERP1 is a secreted glycoprotein with anti-inflammatory activity that is required for full MYX virulence in vivo. The cowpox virus (CPV) serpin SPI-3 (vaccinia virus ORF K2L) is a nonsecreted glycoprotein that blocks cell–cell fusion, independent of serpin activity, and is not required for virulence of vaccinia virus or CPV in mice. Although SPI-3 has only 29% overall identity to SERP1, both serpins have arginine at the P1 position in the reactive center loop, and SPI-3 has a proteinase inhibitory profile strikingly similar to that of SERP1 [Turner, P. C., Baquero, M. T., Yuan, S., Thoennes, S. R., and Moyer, R. W. (2000) Virology 272, 267–280]. To determine whether SPI-3 and SERP1 were functionally equivalent, a CPV variant was constructed where the SPI-3 gene was deleted and replaced with the SERP1 gene regulated by the SPI-3 promoter. Cells infected with CPVΔSPI-3::SERP1 secrete SERP1 and show extensive fusion, suggesting that SERP1 is unable to functionally substitute for SPI-3 in fusion inhibition. In the reciprocal experiment, both copies of SERP1 were deleted from MYX and replaced with SPI-3 under the control of the SERP1 promoter. Cells infected with the MYXΔSERP1::SPI-3 recombinant unexpectedly secreted SPI-3, suggesting either that the cellular secretory pathway is enhanced by MYX or that CPV encodes a protein that prevents SPI-3 secretion. MYXΔSERP1::SPI-3 was as attenuated in rabbits as MYXΔSERP1::lacZ, indicating that SPI-3 cannot substitute for SERP1 in MYX pathogenesis
Characterization of the DNA Replication Module of Bacteriophage A2 and Use of Its Origin of Replication as a Defense against Infection during Milk Fermentation by Lactobacillus casei

AbstractAdjacent to the lysis/lysogeny cassette of the A2 phage genome lies a stretch of over 8 kb, which contains a series of genes probably involved in DNA replication. Fifteen open reading frames (orfs) were identified, 13 of which are encoded on the main coding strand and only two on the complementary strand. Database searches and comparative analyses allowed the identification of an open reading frame (orf455) that shows similarity with DNA helicases and contains a variant zinc-finger motif known from the phage T7 helicase/primase. Orf770 showed similarity to putative plasmid and phage DNA primases. Downstream of orf770 is a noncoding 258-bp region rich in direct and inverted repeats, which specifically binds to proteins whose synthesis is induced during phage infection. When present in a plasmid, this region can direct a partial bacteriophage resistance phenotype due to interference with phage DNA replication, both under laboratory conditions and during milk fermentation. It is deduced that this stretch contains the origin of replication of phage A2
The Brown Algal Virus EsV-1 Particle Contains a Putative Hybrid Histidine Kinase

AbstractThe Ectocarpus siliculosus virus, EsV-1, occurs worldwide in all populations of the filamentous marine brown alga E. siliculosus. We have screened an expression library of EsV-1 restriction fragments and identified a DNA clone with the potential to code for a 52-kDa histidine protein kinase. The derived amino acid sequence includes all homology boxes diagnostic for histidine protein kinases and, in addition, amino acid motifs that are commonly found in response regulators of bacterial two-component signal transduction proteins. Thus, the novel viral protein can be classified as a hybrid histidine protein kinase of a type that has previously been detected in fungi, slime molds, and plants. By using purified antibodies, we found that the protein with its potential kinase activity is located on the outer shell of viral particles. This is the first report on a two-component regulator-like protein in viruses and could provide the basis for speculations with regard to the evolution of EsV-1 and related viruses
Identification and Characterization of a 30K Protein (Ad4E3-30K) Encoded by the E3 Region of Human Adenovirus Type 4

AbstractHuman adenovirus type 4 (Ad4), the sole member of subgroup E, contains an open reading frame in the E3 region predicted to encode a unique 30-kDa protein (named Ad4E3-30K). Ad4E3-30K is predicted to be an integral membrane protein containing an N-terminal signal sequence, a lumenal domain, a transmembrane domain near the C-terminus, and a 37-amino-acid cytoplasmic tail. To determine whether this protein is expressed, rabbit polyclonal antisera were raised against 30K-containing fusion proteins expressed in bacteria. A 30K protein was detected by immunoprecipitation from cell-free translation products and from Ad4-infected A549 cells radiolabeled in the presence of tunicamycin. The protein was detected at only low levels in infected cells. It was not synthesized by a mutant with a large E3 deletion that includes the Ad4E3-30K gene. This mutant grows as well as wild-type Ad4 in culture. Features of Ad4E3-30K were characterized in different transient expression systems. The protein underwent glycosylation by addition of approximately six asparagine-linked oligosaccharides. These glycans were sensitive to endoglycosidase H, indicating that they were either high-mannose or hybrid types, but not complex types, and that the protein did not pass through the Golgi apparatus. Immunofluorescence staining of transfected cells revealed that Ad4E3-30K was localized primarily in the endoplasmic reticulum and nuclear envelope
Ubiquitous Human Adeno-Associated Virus Type 2 Autonomously Replicates in Differentiating Keratinocytes of a Normal Skin Model

AbstractSince its discovery in 1966, adeno-associated virus type 2 (AAV) has been described as a helper-dependent parvovirus. However, in this study we demonstrate that AAV undergoes its complete life cycle, devoid of helper viruses or genotoxic agents, in the organotypic epithelial raft tissue culture system, a model of normal skin. AAV progeny production directly correlated with epithelial differentiation, as nondifferentiating keratinocytes were defective for this activity. Large nuclear virus arrays of particles of approximately 26 nm (parvovirus size) were observed in the granular layers of the raft epithelium by electron microscopy. Additionally, dosage-dependent histologic changes, some of which might be interpreted as cytopathology, were induced in the AAV-infected epithelial tissues. These data suggest a new biological model for AAV; that is, AAV is an epithelial-tropic autonomous parvovirus that can alter normal squamous differentiation
Physical Interaction between Two Varicella Zoster Virus Gene Regulatory Proteins, IE4 and IE62

AbstractTransfection assays demonstrate that the varicella zoster virus (VZV) immediate-early 62 (IE62) protein is a major transactivator of VZV gene expression, whereas a second immediate-early protein, IE4, can act as a major coactivator of transactivation mediated through IE62. To test whether IE62 and IE4 interact physically, we performed several protein–protein interaction assays. Coimmunoprecipitation analyses using VZV-infected cell lysates as well as purified protein mixtures demonstrate that IE62 and IE4 form stable complexes in solution under stringent salt conditions. Enzyme-linked immunosorbent assay protein–protein interaction assays and maltose-binding protein capture assays demonstrate that IE62 binds IE4 in a concentration- and dose-dependent manner. Far Western blot analyses show that IE4 binds to an undermodified form of IE62, and the use of calf intestinal phosphatase and protein kinases suggests that the interaction with IE4 is dependent on the phosphorylation state of IE62. An IE4 binding domain on IE62 has been mapped using a set of truncated IE62 fusion peptides. Collectively, these results imply a direct and specific physical interaction between IE4 and less-phosphorylated forms of IE62. These data have implications for virion assembly, as well as for the regulation of gene expression in VZV-infected cells
H-2d Mice Born to and Reared by HBeAg-Transgenic Mothers Do Not Develop T Cell Tolerance toward the Hepatitis B Virus Core Gene Products

AbstractThe function of the secretory core gene product (HBeAg) of the human hepatitis B virus (HBV) is unknown. It has been proposed that this protein may be passed from the mother to her offspring at the perinatal stage where it might induce immune tolerance. In a previous study we have shown that the murine placenta presents an efficient barrier for the HBe protein and that H-2b mice born to HBeAg-positive transgenic mothers do not develop tolerance of specific cytotoxic T cells. In the present work we demonstrate that transgenic mice expressing high serum levels of HBeAg secrete only small amounts of this protein into their milk and excrete minute amounts of the viral gene product in their urine. Furthermore, it is shown that nontransgenic H-2d mice born to and reared by HBeAg-positive mothers exhibit a reactivity of HBc/eAg-specific CD4+ Th cells and CD8+ cytotoxic T cells comparable to that of normal isogenic control mice. In accordance with this observation the humoral immune responses directed against the HBeAg were comparable between these two groups of animals. This finding indicates that H-2d mice potentially exposed to small amounts of maternal HBeAg transferred by the transplacental, lactogenic, or renal route do not develop tolerance toward the HBV core gene products. These data challenge the hypothesis that a potential function of the HBeAg may be to operate as a tolerogen at the perinatal developmental stage