bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effectiveness of Polyene Antibiotics in Treatment of Transmissible Spongiform Encephalopathy in Transgenic Mice Expressing Syrian Hamster PrP Only in Neurons
Authors: Demaimay Remi , Race Richard , Chesebro Bruce 
Published: December 2019

ABSTRACT
To date very few drugs have favorably influenced the course of transmissible spongiform encephalopathies. In previous studies, the polyene antibiotics amphotericin B (AmB) and MS-8209 prolonged the incubation time in Syrian hamsters of the 263K strain of scrapie, but AmB had no effect against other scrapie strains in Syrian hamsters. In the present experiments using transgenic mice expressing Syrian hamster PrP in neurons only, MS-8209 extended the life spans of animals infected with the 263K strain but not the DY strain. AmB was effective against both 263K and DY and prevented death in 18% of DY-infected animals. The AmB effect against strain 263K was more prominent in mice whose endogenous PrP gene had been inactivated by homologous recombination. It was unclear whether this difference was due to a change in the duration of the disease or to possible interactive effects between the mouse PrP gene and the drugs themselves. The effectiveness of treatment after intracerebral scrapie infection in transgenic mice expressing PrP only in neurons suggested that neurons are important sites of action for these drugs.
Role of Rubella Virus Glycoprotein Domains in Assembly of Virus-Like Particles
Authors: Garbutt Mike , Law Lok Man J. , Chan Honey , Hobman Tom C. 
Published: December 2019

ABSTRACT
Rubella virus is a small enveloped positive-strand RNA virus that assembles on intracellular membranes in a variety of cell types. The virus structural proteins contain all of the information necessary to mediate the assembly of virus-like particles in the Golgi complex. We have recently identified intracellular retention signals within the two viral envelope glycoproteins. E2 contains a Golgi retention signal in its transmembrane domain, whereas a signal for retention in the endoplasmic reticulum has been localized to the transmembrane and cytoplasmic domains of E1 (T. C. Hobman, L. Woodward, and M. G. Farquhar, Mol. Biol. Cell 6:7–20, 1995; T. C. Hobman, H. F. Lemon, and K. Jewell, J. Virol. 71:7670–7680, 1997). In the present study, we have analyzed the role of these retention signals in the assembly of rubella virus-like particles. Deletion or replacement of these domains with analogous regions from other type I membrane glycoproteins resulted in failure of rubella virus-like particles to be secreted from transfected cells. The E1 transmembrane and cytoplasmic domains were not required for targeting of the structural proteins to the Golgi complex and, surprisingly, assembly and budding of virus particles into the lumen of this organelle; however, the resultant particles were not secreted. In contrast, replacement or alteration of the E2 transmembrane or cytoplasmic domain, respectively, abrogated the targeting of the structural proteins to the budding site, and consequently, no virion formation was observed. These results indicate that the transmembrane and cytoplasmic domains of E2 and E1 are required for early and late steps respectively in the viral assembly pathway and that rubella virus morphogenesis is very different from that of the structurally similar alphaviruses.
Sequence and Structural Elements at the 3′ Terminus of Bovine Viral Diarrhea Virus Genomic RNA: Functional Role during RNA Replication
Authors: Yu Haiying , Grassmann Claus W. , Behrens Sven-Erik 
Published: December 2019

ABSTRACTBovine viral diarrhea virus (BVDV), a member of the genusPestivirusin the familyFlaviviridae, has a positive-stranded RNA genome consisting of a single open reading frame and untranslated regions (UTRs) at the 5′ and 3′ ends. Computer modeling suggested the 3′ UTR comprised single-stranded regions as well as stem-loop structures—features that were suspected of being essentially implicated in the viral RNA replication pathway. Employing a subgenomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA replicon (S.-E. Behrens, C. W. Grassmann, H. J. Thiel, G. Meyers, and N. Tautz, J. Virol. 72:2364–2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3′ UTR by experimental means and to investigate the significance of defined RNA motifs for the RNA replication pathway. Enzymatic and chemical structure probing revealed mainly the conserved terminal part (termed 3′C) of the DI9c 3′ UTR containing distinctive RNA motifs, i.e., a stable stem-loop, SL I, near the RNA 3′ terminus and a considerably less stable stem-loop, SL II, that forms the 5′ portion of 3′C. SL I and SL II are separated by a long single-stranded intervening sequence, denoted SS. The 3′-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e.g., with upstream DI9c RNA sequences, were not detected under the experimental conditions used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS motifs do indeed play essential roles during RNA replication. Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as substitution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate. Conversely, SL I stems comprising compensatory base exchanges turned out to support replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c. In summary, these results indicate that RNA structure as well as sequence elements harbored within the 3′C region of the BVDV 3′ UTR create a commoncis-acting element of the replication process. The data further point at possible interaction sites of host and/or viral proteins and thus provide valuable information for future experiments intended to identify and characterize these factors.
Spliced mRNA Encoding the Murine Cytomegalovirus Chemokine Homolog Predicts a β Chemokine of Novel Structure
Authors: MacDonald Margaret R. , Burney Mary W. , Resnick Stuart B. , Virgin Herbert W. 
Published: December 2019

ABSTRACTA viral mRNA of the late kinetic class expressed by murine cytomegalovirus (MCMV) contains an open reading frame (ORF) whose predicted protein, designated MCK-1, has homology to β chemokines (M. R. MacDonald, X.-Y. Li, and H. W. Virgin IV, J. Virol. 71:1671–1678, 1997). The present study analyzed further the structure of the transcript in infected fibroblast cells. A splicing event removed the MCK-1 stop codon, bringing a downstream ORF into frame with the chemokine homolog and demonstrating that the MCK-1 ORF was an exon of a larger gene. The predicted 31.4-kDa protein, designated MCK-2, contains a putative amino-terminal signal sequence and a β chemokine domain, followed by a carboxyl-terminal domain without significant homology to known proteins. Quantitative analysis of mRNA forms in MCMV-infected fibroblast cells at late times after infection indicated that the viral chemokine RNA was predominantly spliced. There was no evidence for expression of RNA encoding either MCK-1 or MCK-2 at immediate early or early times after infection with MCMV. Monoclonal antibodies generated against bacterially expressed MCK-2 recognized multiple proteins in the range of ∼30 to ∼45 kDa in Western blot analysis of MCK-2 expressed in transfected COS cells. The monoclonal antibodies immunoprecipitated a similar group of proteins in transfected COS cells metabolically labeled with radioactive cysteine. Radiolabelled protein of apparent higher molecular mass was immunoprecipitated from culture medium overlying the transfected cells, suggesting that posttranslationally modified MCK-2 can be secreted. Two proteins with apparent molecular mass suggestive of posttranslational modification were detected by Western blot analysis of cells harvested at late times after infection with MCMV. These studies show that MCMV encodes and expresses a β chemokine homolog with a novel predicted structure.
Identification of a Receptor-Binding Pocket on the Envelope Protein of Friend Murine Leukemia Virus
Authors: Davey Robert A. , Zuo Yi , Cunningham James M. 
Published: December 2019

ABSTRACT

Based on previous structural and functional studies, a potential receptor-binding site composed of residues that form a pocket at one end of the two long antiparallel helices in the receptor-binding domain of Friend 57 murine leukemia virus envelope protein (RBD) has been proposed. To test this hypothesis, directed substitutions for residues in the pocket were introduced and consequences for infection and for receptor binding were measured. Receptor binding was measured initially by a sensitive assay based on coexpression of receptor and RBD in
Xenopus
oocytes, and the findings were confirmed by using purified proteins. Three residues that are critical for both binding and infection (S84, D86, and W102), with side chains that extend into the pocket, were identified. Moreover, when mCAT-1 was overexpressed, the infectivity of Fr57-MLV carrying pocket substitutions was partially restored. Substitutions for 18 adjacent residues and 11 other previously unexamined surface-exposed residues outside of the RBD pocket had no detectable effect on function. Taken together, these findings support a model in which the RBD pocket interacts directly with mCAT-1 (likely residues, Y235 and E237) and multiple receptor-envelope complexes are required to form the fusion pore.
Host-Specific Modulation of the Selective Constraints Driving Human Immunodeficiency Virus Type 1
env
Gene Evolution
Authors: Bagnarelli Patrizia , Mazzola Francesca , Menzo Stefano , Montroni Maria , Butini Luca , Clementi Massimo 
Published: December 2019

ABSTRACT

To address the evolution of human immunodeficiency virus type 1 (HIV-1) within a single host, we analyzed the HIV-1 C2-V5
env
regions of both cell-free genomic-RNA- and proviral-DNA-derived clones. Sequential samples were collected over a period of 3 years from six untreated subjects (three typical progressors [TPs] and three slow progressors [SPs], all with a comparable length of infection except one. The evolutionary analysis of the C2-V5
env
sequences performed on 506 molecular clones (253 RNA- and 253 DNA-derived sequences) highlighted a series of differences between TPs and SPs. In particular, (i) clonal sequences from SPs (DNA and RNA) showed lower nucleotide similarity than those from TPs (
P
= 0.0001), (ii) DNA clones from SPs showed higher intra- and intersample nucleotide divergence than those from TPs (
P
< 0.05), (iii) higher host-selective pressure was generally detectable in SPs (DNA and RNA sequences), and (iv) the increase in the genetic distance of DNA and RNA sequences over time was paralleled by an increase in both synonymous (Ks) and nonsynonymous (Ka) substitutions in TPs but only in nonsynonymous substitutions in SPs. Several individual peculiarities of the HIV-1 evolutionary dynamics emerged when the V3, V4, and V5
env
regions of both TPs and SPs were evaluated separately. These peculiarities, probably reflecting host-specific features of selective constraints and their continuous modulation, are documented by the dynamics of Ka/Ks ratios of hypervariable
env
domains.
Modulation of Phosphate Uptake and Amphotropic Murine Leukemia Virus Entry by Posttranslational Modifications of PIT-2
Authors: Rodrigues Pierre , Heard Jean Michel 
Published: December 2019

ABSTRACT
PIT-2 is a type III sodium phosphate cotransporter and the receptor for amphotropic murine leukemia viruses. We have investigated the expression and the functions of a tagged version of PIT-2 in CHO cells. PIT-2 remained equally abundant at the cell surface within 6 h following variation of the phosphate supply. In contrast, the efficiency of phosphate uptake and retrovirus entry was inversely related to the extracellular phosphate concentration, indicating that PIT-2 activities are modulated by posttranslational modifications of cell surface molecules induced by phosphate. Conformational changes of PIT-2 contribute to both activities, as shown by the inhibitory effect of sulfhydryl reagents known as inhibitors of type II cotransporters. A physical association of PIT-2 with actin was demonstrated. Modifications of the actin network were induced by variations of the concentrations of extracellular phosphate, cytochalasin D, or lysophosphatidic acid. They revealed that the formation of actin stress fibers determines the cell surface distribution of PIT-2, the internalization of the receptor in response to virus binding, and the capacity to process retrovirus entry. Thus, the presence of PIT-2 at the cell surface is not sufficient to ensure phosphate transport and susceptibility to amphotropic retrovirus infection. Further activation of cell surface PIT-2 molecules is required for these functions.
Expression and Characterization of a Novel Structural Protein of Human Cytomegalovirus, pUL25
Authors: Battista Maria Concetta , Bergamini Giovanna , Boccuni Maria Cristina , Campanini Fabio , Ripalti Alessandro , Landini Maria Paola 
Published: December 2019

ABSTRACT
Human cytomegalovirus (HCMV) UL25 has recently been found to encode a new structural protein that is present in both virion and defective viral particles (C. J. Baldick and T. Shenk, J. Virol. 70:6097–6105, 1996). In the present work a polyclonal antibody was raised against a prokaryotic pUL25 fusion protein in order to investigate the biosynthesis and localization of the UL25 product (pUL25) during HCMV replication in human fibroblasts. Furthermore, pUL25 was transiently expressed in its native form and fused to the FLAG epitope, in COS7 and U373MG cells, in order to compare the properties of the isolated protein and that produced during infection. Immunoblotting analysis revealed a group of polypeptides, ranging from 80 to 100 kDa, in both transfected and infected cells; in vivo labeling experiments with infected cells demonstrated they are posttranslationally modified by phosphorylation. The transcriptional analysis of the UL25 open reading frame combined with the study of pUL25 biosynthesis showed true late kinetics for this protein in infected human fibroblasts. By indirect immunofluorescence both recombinant and viral pUL25 were detected exclusively in the cytoplasm of transfected or infected cells. Interestingly, pUL25 was shown to localize in typical condensed structures in the perinuclear region as already observed for other HCMV tegument proteins. Colocalization of ppUL99 in the same vacuoles suggests that these structure are endosomal cisternae, which are proposed to be a preferential site of viral particle envelopment. Our data suggest that pUL25 is most likely a novel tegument protein and possibly plays a key role in the process of envelopment.
Highly Diverse Intergenic Regions of the Paramyxovirus Simian Virus 5 Cooperate with the Gene End U Tract in Viral Transcription Termination and Can Influence Reinitiation at a Downstream Gene
Authors: Rassa John C. , Parks Griffith D. 
Published: December 2019

ABSTRACT
A dicistronic minigenome containing the M-F gene junction was used to determine the role of the simian virus 5 (SV5) intergenic regions in transcription. The M-F junction differs from the other SV5 junctions by having a short M gene end U tract of only four residues (U4 tract) and a 22-base M-F intergenic sequence between the M gene end and F gene start site. Replacing the 22-base M-F intergenic region with nonviral sequences resulted in a minigenome template (Rep 22) that was defective in termination at the end of the M gene. Efficient M gene termination could be restored to the mutant Rep 22 template in either of two ways: by increasing the U tract length from four to six residues or by restoring a G residue immediately downstream of the wild-type (WT) U4 tract. In a dicistronic SH-HN minigenome, a U4-G combination was functionally equivalent to the naturally occurring SH U6-A gene end in directing SH transcription termination. In addition to affecting termination, the M-F intergenic region also influenced polymerase reinitiation. In the context of the WT U4-G M gene end, substituting nonviral sequences into the M-F intergenic region had a differential effect on F gene reinitiation, where some but not all nonviral sequences inhibited reinitiation. The inhibition of F gene reinitiation correlated with foreign sequences having a high C content. Deleting 6 bases or inserting 18 additional nucleotides into the middle of the 22-base M-F intergenic segment did not influence M gene termination or F gene reinitiation, indicating that M-F intergenic length per se is not a important factor modulating the SV5 polymerase activity. Our results suggest that the sequence diversity at an SV5 gene junction reflects specific combinations which may differentially affect SV5 gene expression and provide an additional level of transcriptional control beyond that which results from the distance of a gene from the 3′ end promoter.
Cooperative Interaction of CI Protein Regulates Lysogeny of
Lactobacillus casei
by Bacteriophage A2
Authors: García Pilar , Ladero Victor , Alonso Juan C. , Suárez Juan E. 
Published: December 2019

ABSTRACT

The temperate bacteriophage A2 forms stable lysogens in
Lactobacillus casei
. The A2-encoded
cI
product (CI), which is responsible for maintaining the A2 prophage in the lysogenic state, has been purified. The CI protein, which is a monomer of 25.3 kDa in solution, specifically binds to a 153-bp DNA fragment that contains two divergent promoters, P
L
and P
R
. These promoters mediate transcription from
cI
and a putative
cro
, respectively. Three similar, although not identical, 20-bp inverted repeated DNA segments (operator sites O
1
, O
2
, and O
3
) were found in this segment. CI selectively interacts with O
1
, which is placed downstream from the transcription start point of the
cro
gene, and with O
2
and O
3
, which overlap with the −35 region of the two promoters. Using a heterologous RNA polymerase, we have determined the transcription start points of P
L
and P
R
. CI exerts a negative effect on the in vitro transcription of P
R
by repositioning the RNA polymerase in a concentration-dependent manner. CI, when bound to O
1
and O
2
, enhances the positioning of the RNA polymerase with the P
L
promoter. Our data indicate that the CI protein regulates the lytic and lysogenic pathways of the A2 phage.
In Vitro Recoating of Reovirus Cores with Baculovirus-Expressed Outer-Capsid Proteins μ1 and ς3
Authors: Chandran Kartik , Walker Stephen B. , Chen Ya , Contreras Carlo M. , Schiff Leslie A. , Baker Timothy S. , Nibert Max L. 
Published: December 2019

ABSTRACT

Reovirus outer-capsid proteins μ1, ς3, and ς1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed μ1 and ς3 onto purified cores that lack μ1, ς3, and ς1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein ς1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of μ1 and ς3 onto cores had induced rearrangement of the pentameric λ2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH
4
Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the ς1 protein. To examine the utility of r-cores for genetic dissections of μ1 functions in reovirus entry, we generated r-cores containing a mutant form of μ1 that had been engineered to resist cleavage at the δ:φ junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in δ:φ cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH
4
Cl, providing new evidence that this cleavage is dispensable for productive infection.
Complete Sequence of Enzootic Nasal Tumor Virus, a Retrovirus Associated with Transmissible Intranasal Tumors of Sheep
Authors: Cousens Christina , Minguijon Esmeralda , Dalziel Robert G. , Ortin Aurora , Garcia Mercedes , Park Jane , Gonzalez Lorenzo , Sharp J. Michael , de las Heras Marcelo 
Published: December 2019

ABSTRACT
The sequence of the complete genome of ovine enzootic nasal tumor virus, an exogenous retrovirus associated exclusively with contagious intranasal tumors of sheep, was determined. The genome is 7,434 nucleotides long and exhibits a genetic organization characteristic of type B and D oncoviruses. Enzootic nasal tumor virus is closely related to the Jaagsiekte sheep retrovirus and to sheep endogenous retroviruses.
Jaagsiekte Retrovirus Is Widely Distributed both in T and B Lymphocytes and in Mononuclear Phagocytes of Sheep with Naturally and Experimentally Acquired Pulmonary Adenomatosis
Authors: Holland Martin J. , Palmarini Massimo , Garcia-Goti Mercedes , Gonzalez Lorenzo , McKendrick Iain , de las Heras Marcelo , Sharp J. Michael 
Published: December 2019

ABSTRACTJaagsiekte sheep retrovirus (JSRV) is a type D retrovirus specifically associated with a contagious lung tumor of sheep, sheep pulmonary adenomatosis (SPA). JSRV replicates actively in the transformed epithelial cells of the lung, and JSRV DNA and RNA have been detected in lymphoid tissues of naturally affected animals. To determine the lymphoid target cells of JSRV, CD4+T cells, CD8+T cells, B lymphocytes, and adherent cell (macrophage/monocyte) populations were isolated from the mediastinal lymph nodes of naturally affected sheep and lambs inoculated with JSRV. Cells were enriched to high purity and then analyzed for JSRV proviral DNA by heminested PCR, and the proviral burden was quantitated by limiting dilution analysis. JSRV proviral DNA was found in all subsets examined but not in appropriate negative controls. In sheep naturally affected with SPA, JSRV proviral burden was greatest in the adherent cell population. In the nonadherent lymphocyte population, surface immunoglobulin-positive B cells contained the greatest proviral burden, while CD4+and CD8+T cells contained the lowest levels of JSRV proviral DNA. In most of the cases (5 of 8), provirus also could be detected in the peripheral blood mononuclear cell (PBMC) population. A kinetic study of JSRV infection in the mediastinal lymphocyte population of newborn lambs inoculated with JSRV found that JSRV proviral DNA could be detected as early as 7 days postinoculation before the onset of pulmonary adenomatosis, although the proviral burden was greatly reduced compared to adult natural cases. This was reflected in the levels found in PBMC since proviral DNA was detected in 2 of 13 animals. At the early time points studied (7 to 28 days postinoculation) no one subset was preferentially infected. These data indicate that JSRV can infect lymphoid and phagocytic mononuclear cells of sheep and that dissemination precedes tumor formation. Infection of lymphoid tissue, therefore, may play an important role in the pathogenesis of SPA.