pinecone/core-2020-05-10-deduplication huggingface dataset:
Stable Expression of the Reovirus μ2 Protein in Mouse L Cells Complements the Growth of a Reovirus ts Mutant with a Defect in Its M1 Gene

AbstractReovirus μ2 protein was constitutively expressed in mammalian cells transfected with dicistronic constructs in which the reovirus M1 gene and the selectable neomycin-resistant gene (neo) were both driven by the same phosphoglycerate kinase promoter. Translation of neo was initiated with the cap-independent translation initiation element from encephalomyocarditis virus. Expression of μ2 protein was detected by μ2-specific antibody produced through immunization of rabbits with Trp-E-μ2 fusion proteins expressed inEscherichia coli.The expression levels of μ2 proteins of serotype 1 (T1) and serotype 3 (T3) were different and varied in different mouse cell lines with T1 being expressed more efficiently than T3. μ2-expressing L929 cell lines generated with the dicistronic constructs were highly stable. Inclusion of the transforming fragment of bovine papillomavirus in the dicistronic construct lead to higher levels of μ2 expression that were less stable and thus decreased on continued cell culture. The μ2 protein expressed in transfectants was authentic as shown by peptide mapping comparison with μ2 protein from reovirus-infected cells and that fromin vitrotranscription and translation of the M1 gene. It was further shown that the μ2 protein expressed in a stable L929 cell line complemented the growth of the reovirus tsH11.2 mutant with a defect in its M1 gene. It is concluded that the μ2 protein stably expressed by transfection is functionally equivalent to μ2 protein expressed by reovirus
An Altered Form of Apolipoprotein H Binds Hepatitis B Virus Surface Antigen Most Efficiently

AbstractUsing recombinant (r)HBsAg as a ligand, we previously found a 46-kDa human plasma protein capable of specific binding, and identified this protein as apolipoprotein H (apo H). Apo H is able to bind to rHBsAg containing only the small S protein, in both ligand blot and enzyme immunoassay systems (H. Mehdi, M. J. Kaplan, F. Y. Anlar., X. Yang, R. Bayer, K. Sutherland, and M. E. Peeples,J. Virol.68, 2415–2424, 1994). Apo H is a plasma glycoprotein, some of which is associated with lipoproteins, particularly chylomicrons and high-density lipoproteins (HDL). During normal lipid trafficking in the bloodstream, chylomicrons and HDL are targeted to the hepatocyte, the primary host cell for HBV, for degradation. In this report the method of apo H presentation was examined. rHBsAg bound to apo H very poorly if the apo H was coated directly on a microtiter well, or if it was presented in a soluble form. Binding was 100-fold more efficient when apo H was presented as a complex with monoclonal antibody (MAb) P2D4. These results suggest that binding to this MAb alters apo H, making it highly reactive with rHBsAg. Apo H binding to rHBsAg is not dependent on divalent cations and is optimal at pH 6.5–8.0. Removal of lipids from rHBsAg resulted in denaturation, preventing analysis of binding activity. Removal of sialic acid or complete removal of N-linked carbohydrates from apo H did not change its ability to bind rHBsAg, indicating that apo H carbohydrates are not involved in rHBsAg binding. Likewise, chemical modification of the arginine residues of apo H had no effect on binding. However, chemical modification of as few as three of the 29 lysine residues of apo H destroyed binding, indicating that one or a few lysines in apo H are involved in rHBsAg binding
Nuclear Domain 10 as Preexisting Potential Replication Start Sites of Herpes Simplex Virus Type-1

AbstractThe herpes simplex virus type 1 (HSV-1) nuclear replication cycle begins at localized sites, but it has remained unclear whether these sites are associated with any defined nuclear structure. We have previously shown that during infection, the HSV-1 immediate-early protein ICP0 dispersed proteins associated with ND10, nuclear sites that contain high concentrations of PML and other potentially regulatory proteins and correspond to the ultrastructurally defined nuclear bodies. Usingin situhybridization and immunohistochemical techniques, we found that ICP0 mutants of HSV-1 replicate in the close proximity with ND10, but increasing replication sites develop away from these nuclear structures. Input wild-type HSV-1 DNA was found preferentially adjacent to ND10 before ICP0 modified these nuclear structures and did not colocalize with ICP8 containing so-called prereplication sites. The sites where HSV-1 can begin replication then need to be redefined as preexisting potential replication sites. Viral RNA was also found associated with ND10 before early protein synthesis (ICP8), suggesting that input virus genomes are deposited at ND10 before they start replication. The deposition of input viral DNA at ND10 is virus gene expression independent, probably indicating cell regulation of this process. Taken together, these data demonstrate that some very early processes of the nuclear viral replication cycle happen in close proximity or at the periphery of ND10. The localization of input HSV-1 to ND10 represents a new host–virus interaction and provides an unexpected functional property for this large nuclear site
Complementation of M Gene Mutants of Vesicular Stomatitis Virus by Plasmid-Derived M Protein Converts Spherical Extracellular Particles into Native Bullet Shapes

AbstractThe matrix (M) protein of vesicular stomatitis virus (VSV) binds the nucleocapsid to the cytoplasmic surface of the host plasma membrane during virus assembly by budding. It also condenses the nucleocapsid into a tightly coiled nucleocapsid–M protein complex that appears to give the virion its bullet-like shape. As described here, temperature-sensitive (ts) M mutants produced two classes of membrane-containing extracellular particles at the nonpermissive temperature. These could be distinguished by sedimentation in sucrose gradients and by electron microscopy. One class contained nucleocapsids and envelope glycoprotein, but very little M protein. The other class was devoid of nucleocapsids. Most of these particles were spherical or pleiomorphic in shape as determined by electron microscopy. Expression of wild-type (wt) M protein from plasmid DNA using the vaccinia/T7 virus system did not enhance the incorporation of nucleocapsids into extracellular particles from cells coinfected with the ts M mutants but did enhance the incorporation of M protein into these particles. Electron microscopy showed that wt M protein served to impart the bullet-like shape typical of VSV virions to what would otherwise be spherical or pleiomorphic virus-like particles. These data suggest that there are two distinct processes in VSV envelope biogenesis. One process involves envelopment of the nucleocapsid and can be accomplished by the ts M mutants at the nonpermissive temperature, albeit at a low level compared to wt VSV. The other process involves conversion of virion components into the bullet-like shape and requires a function provided by wt M protein
Functional Analysis of dsRNAs (L1, L3, L5, and M2) Associated with Isometric 34-nm Virions ofAgaricus bisporus(White Button Mushroom)

AbstractcDNA clones of dsRNAs associated with La France disease ofAgaricus bisporuswere isolated. Clones corresponding to L1 and L5 dsRNAs were sequenced. The deduced amino acid sequence of L1 dsRNA (1078 amino acids,Mr121K) showed significant homology with RNA-dependent RNA polymerases of other dsRNA viruses. The deduced amino acid sequence of L5 dsRNA (724 amino acids,Mr82K) showed no homology with known proteins. Amino acid sequences of tryptic digests of three virion-associated proteins were determined. The 34-nm virion-associated protein ofMr115K was encoded by the L1 dsRNA, thus identifying this protein as the RNA-dependent RNA polymerase. The virion-associated protein ofMr90K was encoded by the previously sequenced L3 dsRNA. A cDNA clone of the previously sequenced M2 dsRNA was expressed inEscherichia coliand antibodies raised against this protein reacted only with a protein present in the cytoplasm of diseasedA. bisporusfruit bodies but not in the 34-nm virions
Identification and Analysis of anAutographa californicaNuclear Polyhedrosis Virus Structural Protein of the Occlusion-Derived Virus Envelope: ODV-E56

AbstractAnAutographa californicanuclear polyhedrosis virus gene encoding an occlusion-derived virus (ODV) envelope protein of 56 kDa was identified and sequenced. Transcription initiates from a conserved baculovirus late motif (ATAAG) with transcripts detected from 16 through 72 hr p.i. The protein is detected in infected cell extracts from 36 hr p.i. Western blot assay of ODV, BV, viral envelope, and nucleocapsid preparations coupled with immunoelectron microscopy reveal that this protein localizes to the ODV envelope. This protein is named ODV-E56 to identify its viral origin, envelope location, and apparent molecular weight. ODV-E56 is enriched in viral induced intranuclear microvesicles as determined by immunogold labeling. A mutant was constructed with the C-terminal portion of the protein replaced with β-galactosidase. The fusion protein, E56-β-gal, locates to the viral nucleocapsids and not to the ODV envelope or intranuclear microvesicles. This suggests that the signals necessary for transport and/or retention into these structures lies within the C-terminal portion of ODV-E56. Additionally, both ODV-E56 and E56-β-gal are enriched in electron dense regions that cluster around the inner nuclear membrane and within the nucleoplasm
Isolation and Characterization of Herpes Simplex Virus Type 1 Mutants Defective in the UL6 Gene

AbstractPrevious studies have shown that the protein encoded by herpes simplex virus type 1 (HSV-1) gene UL6 is required for processing and packaging of replicated viral DNA and is a minor component of virions and capsids. In this report, we describe the construction of UL6−HSV-1 mutants with a disrupted UL6 gene using complementing cells and show that they fail to synthesize the UL6 protein or produce infectious virus in noncomplementing cells. The mutants synthesized but failed to process and encapsidate viral DNA and accumulated only immature capsids which lacked the UL6 protein. Immunofluorescence analysis showed that the UL6 protein, when expressed transiently in transfected cells in the absence of other HSV-1 proteins, is localized exclusively to the nucleus. We also investigated an HSV-1 mutant with a defect in gene UL33, the product of which is also thought to be involved in viral DNA processing and packaging. The phenotype of this mutant on noncomplementing cells with regard to failure to process and encapsidate viral DNA, accumulation of immature capsids, and inability to produce infectious virus was the same as that of UL6−viruses. This mutant, however, produced capsids containing the UL6 protein, indicating that association of the UL6 protein with the capsid is independent of the UL33 protein
Adeno-Associated Virus Type 2 Binds to a 150-Kilodalton Cell Membrane Glycoprotein

AbstractEarly events, including the identification of the cellular receptor, have not yet been described for adeno-associated virus (AAV) infection. In this study, the binding characteristics of AAV-2 to human cells were examined in two different assays. In a liquid-phase assay, in which binding of biotinylated virus to cells in suspension was measured, AAV-2 showed specific binding to four different permissive cell lines: HeLa S3, 293, D6, and KB cells. In contrast, AAV-2 binding to erythrocytes or to the nonpermissive cell line UT-7/Epo was negligible. AAV-2 binding showed saturation kinetics. Both binding and infectivity of AAV-2 were abolished by trypsin treatment of cells, with significant recovery of bindings after 8 hr of culture, suggesting that virus attachment occurs through a protein that can be regenerated on the cell surface. In a second, virus overlay assay, we assessed binding of [35S]methionine-labeled AAV-2 to membrane proteins that had been transferred to nitrocellulose after electrophoretic separation. In this assay, virus attachment was shown to a 150-kDa protein. This protein was present in membranes from the AAV-2 permissive cell lines but not detected in membranes from erythrocytes or UT-7/Epo cells. Enzymatic deglycosylation studies suggested that N-linked glycans are required for AAV-2 binding. A 150-kDa glycoprotein might serve as the cellular receptor for AAV-2
Characterization of Human Adenovirus Proteinase Activity in Disrupted Virus Particles

AbstractVirus-coded proteinases are attractive targets for antiviral therapy; however, lack of quick, sensitive, quantitative, and selective assays for enzyme activity makes it difficult to characterize these proteinases and to screen large numbers of potential inhibitors. Here we describe new substrates for the adenovirus proteinase, fluorogenic Rhodamine-based substrates containing tetrapeptides corresponding to sequences cleaved in adenovirus precursor proteins. Proteinase activity in as few as 109disrupted virions could be quantitatively detected in a 30-min assay. With the substrate (Leu-Arg-Gly-Gly-NH)2–Rhodamine, theKmwas 1.4 μM,and theVmaxwas 3.24 pmol substrate hydrolyzed/sec/pmol virus. Enzyme activity was stimulated by dithiothreitol and inhibited by several serine-specific as well as cysteine-specific proteinase inhibitors. In a thiol protection experiment, the virion enzyme was shown to have a cysteine residue with an unusually low pKa, a pKasimilar to that of the active-site nucleophile of the cysteine proteinase papain. The curve ofVmaxas a function of pH is unlike the curve from papain and implied that there are at least three ionizable groups whose protonation state can affect catalysis—one with a pKaof 6.2, another with a pKaof 7.2, and a third with a pKaof 8.3
High Activated and Memory Cytotoxic T-cell Responses to HTLV-1 in Healthy Carriers and Patients with Tropical Spastic Paraparesis

AbstractThe cytotoxic T-lymphocyte (CTL) response to HTLV-1 is directed mainly against the Tax protein. Circulating, activated Tax-specific CTL can be found in a majority of healthy carriers and patients with the HTLV-1-associated disease tropical spastic paraparesis (HAM/TSP). In this study we present data on the Tax-specific CTL response of 26 HTLV-1 carriers, including 10 newly recruited subjects. Rex-specific CTL were not found in any subjects investigated. Activated and memory CTL responses were determined separately in 4 healthy carriers, 3 HAM/TSP patients, and 1 “seronegative HAM/TSP.” In all subjects, the mean frequency of peptide-specific memory cells per epitope (1/1307) was high. There was no significant difference in mean memory CTL frequency per epitope or in the proportion of subjects with activated CTL between healthy carriers and HAM/TSP patients. One individual with HAM/TSP had an unusually high frequency response to two peptides, suggesting immunodominance of epitope recognition in this individual. We conclude that the magnitude and components of the HTLV-1-specific CTL response do not differ between healthy carriers and HAM/TSP patients. These data do not support a specific CTL-mediated component in the pathogenesis of HAM/TSP
Interaction between Cauliflower Mosaic Virus Inclusion Body Protein and Capsid Protein: Implications for Viral Assembly

AbstractThe cauliflower mosaic virus (CaMV) inclusion body protein (pVI) is able to specifically interact with the viral capsid precursor protein (pIV). By using the yeast two-hybrid system and a blot assay, the pIV region required for the recognition of pVI was mapped to the lysine-rich domain. This region of only 48 amino acids when fused to dihydrofolate reductase (DHFR) mediated pVI and DNA bindingin vitro.Competition experiments confirmed that pVI and DNA bind to the same region of pIV. Since pVI is absent from the mature virus, models are discussed in which pVI plays an accessory role in CaMV assembly, in addition to its function in transactivating translation
Development of anin VitromRNA Degradation Assay Utilizing Extracts from HIV-1- and SIV-Infected Cells

AbstractWe previously demonstrated that cellular mRNAs are degraded in CD4 positive lymphocytes infected by the human immunodeficiency virus, HIV-1, but not in cells infected by the simian lentivirus, SIV. To begin to define the molecular mechanisms underlying this RNA degradation, we have established anin vitroRNA degradation assay utilizing extracts from both infected and uninfected cells. We found thatin vitrotranscribed,32P-radiolabeled actin RNA was degraded in extracts prepared from CEM, CEMx174, and C8166 cells which were infected with HIV-1. Minimal actin RNA degradation was observed in extracts prepared from uninfected cells. Similarly little degradation was observed in cell-free extracts prepared from SIV-infected cells. To determine if viral RNA sequences could impart enhanced stability to cellular RNAs in ourin vitroassay, we prepared radiolabeled RNAs that contained selected viral RNA determinants. One such RNA contained the HIV-1-specific TAR (transactivating region) sequence (nucleotides 1–111) appended to a reporter CAT RNA. Like the cellular actin RNA, these TARCAT RNAs were degraded in HIV-1-infected cell extracts, but not in extracts from uninfected cells or extracts prepared from SIV-infected cells. In contrast, an RNA containing only authentic HIV-1 sequences comprising TAR and gag sequences was more stable than actin RNA in HIV-1-infected extracts. These results, taken together, suggest that thein vitroassay reproduces events that occurin vivoand provide a starting point for identifying the factors responsible for cellular RNA degradation in HIV-1-infected cells
Lymphocyte Activators Elicit Bovine Leukemia Virus Expression Differently as Asymptomatic Infection Progresses

AbstractSince bovine leukemia virus (BLV) replicates in B-lymphocytes, viral expression and production should respond to agents activating these cells. We asked whether synthesis of BLV capsid (CA) protein and production of infectious virus would increase when peripheral blood mononuclear cells (PBMCs) from infected sheep were stimulated in short-term culture with lipopolysaccharide (LPS), a polyclonal activator of B-cells, and compared its effects with those of phytohemagglutinin (PHA), a lymphocyte activator known to increase BLV expression. LPS treatment of PBMCs from asymptomatic sheep that had been infected for 1–4 years increased the number of cells synthesizing CA protein, the amount of CA protein per cell, and the number of PBMCs acting as infectious centers. LPS derived from several different microbes was effective. During the ensuing 4 years of asymptomatic infection, the number of PBMCs expressing virus under minimal stimulation increased for each animal. The ability of LPS to recruit additional cells to express CA protein remained constant or decreased in magnitude, yet at the same time, lower concentrations of LPS were required to elicit a maximal effect. This suggests that the cellular pathways affected by LPS are endogenously more activated as infection progresses. PHA initially stimulated fewer cells to synthesize BLV CA protein than LPS did although the amount of CA protein synthesized per cell was greater with PHA. As infection progressed, PHA surpassed LPS in the numbers of PBMCs induced to express CA protein. This suggests that the cellular pathways affected by PHA become more responsive to its effects as infection progresses. LPS increased CA expression early and transiently during culture whereas the PHA-mediated increase continued to develop for several days. Thus, LPS increases BLV expression but does so differently than PHA. Moreover, these longitudinal results show that the activation state of BLV-infected cells changes as asymptomatic infection progresses
Targeting of Retroviral Integrase by Fusion to a Heterologous DNA Binding Domain:In VitroActivities and Incorporation of a Fusion Protein into Viral Particles

AbstractRetroviral DNA integration is catalyzed by a viral protein, the integrase (IN). IN recognizes sequences at the viral DNA ends, specifically nicks these ends (theprocessingreaction), and inserts them into host DNA (thejoiningreaction). The mechanism by which host DNA integration sites are selected is unknown, although it is clear that many regions are accessible to the retroviral integration machinery. To investigate integration site selection, and to possibly influence this process, we have used a model system in which the avian sarcoma virus (ASV) IN, and segments thereof, have been fused to theEscherichia coliLexA repressor protein DNA binding domain (DBD). We provide evidence that a variety of such IN-LexA DBD fusion proteins can be directed to cognatelexA operatorsin vitro.Binding of precise N- and C-terminal fusions proteins to the operators is associated with a significant increase in the joining of viral DNA to sites immediately adjacent to thelexA operator region. We also carried out domain exchange and deletion experiments which revealed that the highly conserved “zinc-finger” domain of ASV IN is not essential for processing of viral DNA ends or joining of viral DNA ends to DNA target sequences. Last, the C-terminal fusion was engineered into the viral genome and we show that this protein can be incorporated into viral particles. Our results suggest that it might be possible to augment IN functionin vivothrough a heterologous domain. These observations have implications for retroviral-mediated gene therapy
The Infectious Bronchitis Virus Nucleocapsid Protein Binds RNA Sequences in the 3′ Terminus of the Genome

AbstractThe infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a bacterial fusion protein which differed from the native protein only in the addition of six amino terminus histidine residues. Using RNA overlay protein blot assays, the recombinant protein was shown to bind to RNA fragments specific for the positive sense 3′ noncoding end of the IBV genome. At greater concentrations of sodium chloride, the native and fusion nucleocapsid proteins similarly bound to G RNA, representing the terminal 1805 3′ nt of the genome, whereas bovine serum albumin and allantoic fluid protein did not bind to labeled G RNA. Competitive gel shift assays with labeled G RNA indicated that the protein interacted with several unlabeled RNA representing sequences at the 3′ noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the small segment cDNA also inhibited the interaction with IBV G RNA to approximately the same extent as homologous unlabeled G RNA, whereas reactions with bovine liver RNA and yeast tRNA were considerably weaker. Whereas yeast tRNA did not inhibit the interaction with the labeled large G RNA, interactions of the fusion protein with EF, a region from 78 to 217 nt from the 3′ terminus of the IBV genome, were also apparently weaker than interactions with fragment CD which consisted of the 3′ terminal 155 nt. On a molar basis, the latter interacted in an identical nature to a RNA consisting of CD and an additional 1053 nt of plasmid sequences. Compared to bovine liver RNA, unlabeled G specifically inhibited binding to the two smaller labeled IBV fragments in gel shift assays. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present on the genome, and may represent a common mechanism used by similar viral nucleoproteins whose functions depend on binding to RNA