bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Null Mutation in the UL36 Gene of Herpes Simplex Virus Type 1 Results in Accumulation of Unenveloped DNA-Filled Capsids in the Cytoplasm of Infected Cells
Authors: Desai Prashant J. 
Published: July 2002

ABSTRACT
The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designated VP1/2, which is also a component of the virion tegument. A null mutation was generated in the UL36 gene to elucidate its role in the virus life cycle. Since the UL36 gene specifies an essential function, complementing cell lines transformed for sequences encoding the UL36 ORF were made. A mutant virus, designated KΔUL36, that encodes a null mutation in the UL36 gene was isolated and propagated in these cell lines. When noncomplementing cells infected with KΔUL36 were analyzed, both terminal genomic DNA fragments and DNA-containing capsids (C capsids) were detected; therefore, UL36 is not required for cleavage or packaging of DNA. Sedimentation analysis of lysates from mutant-infected cells revealed the presence of particles that have the physical characteristics of C capsids. In agreement with this, polypeptide profiles of the mutant particles revealed an absence of the major envelope and tegument components. Ultrastructural analysis revealed the presence of numerous unenveloped DNA containing capsids in the cytoplasm of KΔUL36-infected cells. The UL36 mutant particles were tagged with the VP26-green fluorescent protein marker, and their movement was monitored in living cells. In KΔUL36-infected cells, extensive particulate fluorescence corresponding to the capsid particles was observed throughout the cytosol. Accumulation of fluorescence at the plasma membrane which indicated maturation and egress of virions was observed in wild-type-infected cells but was absent in KΔUL36-infected cells. In the absence of UL36 function, DNA-filled capsids are produced; these capsids enter the cytosol after traversing the nuclear envelope and do not mature into enveloped virus. The maturation and egress of the UL36 mutant particles are abrogated, possibly due to a late function of this complex polypeptide, i.e., to target capsids to the correct maturation pathway.
Affinity-Tagged Miniprion Derivatives Spontaneously Adopt Protease-Resistant Conformations
Authors: Supattapone Surachai , Nguyen Hoang-Oanh B. , Muramoto Tamaki , Cohen Fred E. , DeArmond Stephen J. , Prusiner Stanley B. , Scott Michael 
Published: July 2002

ABSTRACT

An abridged PrP molecule of 106 amino acids designated PrP106 can form infectious miniprions in transgenic (Tg) mice (29). Addition of six-histidine (His
6
) affinity tags to selective sites within PrP106 resulted unexpectedly in new PrP proteins that spontaneously adopted protease-resistant conformations when expressed in neuroblastoma cells and Tg mice. Acquisition of protease resistance depended on the length, charge, and placement of the affinity tag. Introduction of the disease-linked mutation E200K into the sequence of PrP106(140/6His) increased the recovery of protease-resistant PrP fivefold, whereas introduction of the mutations C213A and Δ214–220 did not affect the recovery of protease-resistant PrP. Treatment of cultured cells expressing affinity-tagged PrP106 mutants with polypropyleneimine dendrimer rendered these proteins sensitive to protease digestion in a manner similar to wild-type PrP
Sc
. We conclude that certain affinity-tagged PrP106 proteins spontaneously fold into conformations partially resembling, yet distinct from, wild-type PrP
Sc
. These proteins might be useful tools in the identification of new disease-causing mutations as well as for screening compounds for therapeutic efficacy.
Avian Endogenous Retrovirus EAV-HP Shares Regions of Identity with Avian Leukosis Virus Subgroup J and the Avian Retrotransposon ART-CH
Authors: Sacco M. A. , Flannery D. M. J. , Howes K. , Venugopal K. 
Published: July 2002

ABSTRACT

The existence of novel endogenous retrovirus elements in the chicken genome, designated EAV-HP, with close sequence identity to the
env
gene of avian leukosis virus (ALV) subgroup J has been reported (L. M. Smith, A. A. Toye, K. Howes, N. Bumstead, L. N. Payne, and K. Venugopal, J. Gen. Virol. 80:261–268, 1999). To resolve the genome structure of these retroviral elements, we have determined the complete sequence of two proviral clones of EAV-HP from a line N chicken genomic DNA yeast artificial chromosome library and from a meat-type chicken line 21 lambda library. The EAV-HP sequences from the two lines were 98% identical and had a typical provirus structure. The two EAV-HP clones showed identical large deletions spanning part of the
gag
, the entire
pol
, and part of the
env
genes. The
env
region of the EAV-HP clones was 97% identical to the
env
sequence of HPRS-103, the prototype subgroup J ALV. The 5′ region of EAV-HP comprising the R and U5 regions of the long terminal repeat (LTR), the untranslated leader, and the 5′ end of the putative
gag
region were 97% identical to the avian retrotransposon sequence, ART-CH. The remaining
gag
sequence shared less than 60% identity with other ALV sequences. The U3 region of the LTR was distinct from those of other retroviruses but contained some of the conserved motifs required for functioning as a promoter. To examine the ability of this endogenous retroviral LTR to function as a transcriptional promoter, the EAV-HP and HPRS-103 LTR U3 regions were compared in a luciferase reporter gene assay. The low luciferase activity detected with the EAV-HP LTR U3 constructs, at levels close to those observed for a control vector lacking the promoter or enhancer elements, suggested that these elements function as a weak promoter, possibly accounting for their low expression levels in chicken embryos.
The RNA Helicase and Nucleotide Triphosphatase Activities of the Bovine Viral Diarrhea Virus NS3 Protein Are Essential for Viral Replication
Authors: Gu Baohua , Liu Changbao , Lin-Goerke Juili , Maley Derrick R. , Gutshall Lester L. , Feltenberger Cynthia A. , Del Vecchio Alfred M. 
Published: July 2002

ABSTRACT

Helicase/nucleoside triphosphatase (NTPase) motifs have been identified in many RNA virus genomes. Similarly, all the members of the
Flaviviridae
family contain conserved helicase/NTPase motifs in their homologous NS3 proteins. Although this suggests that this activity plays a critical role in the viral life cycle, the precise role of the helicase/NTPase in virus replication or whether it is essential for virus replication is still unknown. To determine the role of the NS3 helicase/NTPase in the viral life cycle, deletion and point mutations in the helicase/NTPase motifs of the bovine viral diarrhea virus (BVDV) (NADL strain) NS3 protein designed to abolish either helicase activity alone (motif II, DEY
H
to DEY
A
) or both NTPase and helicase activity (motif I, G
K
T to G
A
T and deletion of motif VI) were generated. The C-terminal domain of NS3 (BVDV amino acids 1854 to 2362) of these mutants and wild type was expressed in bacteria, purified, and assayed for RNA helicase and ATPase activity. These mutations behaved as predicted with respect to RNA helicase and NTPase activities in vitro. When engineered back into an infectious cDNA for BVDV (NADL strain), point mutations in either the GKT or DEYH motif or deletion of motif VI yielded RNA transcripts that no longer produced infectious virus upon transfection of EBTr cells. Further analysis indicated that these mutants did not synthesize minus-strand RNA. These findings represent the first report unequivocably demonstrating that helicase activity is essential for minus-strand synthesis.
JC Virus Enters Human Glial Cells by Clathrin-Dependent Receptor-Mediated Endocytosis
Authors: Pho M. T. , Ashok A. , Atwood Walter J. 
Published: July 2002

ABSTRACT
The human polyomavirus JC virus (JCV) is the etiologic agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs predominantly in immunosuppressed patients and has increased dramatically as a result of the AIDS pandemic. The major target cell of JCV infection and lytic replication in the CNS is the oligodendrocyte. The mechanisms by which JCV initiates and establishes infection of these glial cells are not understood. The initial interaction between JCV and glial cells involves virus binding to N-linked glycoproteins containing terminal α(2-6)-linked sialic acids. The subsequent steps of entry and targeting of the viral genome to the nucleus have not been described. In this report, we compare the kinetics and mechanisms of infectious entry of JCV into human glial cells with that of the related polyomavirus, simian virus 40 (SV40). We demonstrate that JCV, unlike SV40, enters glial cells by receptor-mediated clathrin-dependent endocytosis.
Inhibition of CD3/CD28-Mediated Activation of the MEK/ERK Signaling Pathway Represses Replication of X4 but Not R5 Human Immunodeficiency Virus Type 1 in Peripheral Blood CD4+T Lymphocytes
Authors: Popik Waldemar , Pitha Paula M. 
Published: July 2002

ABSTRACTBinding of human immunodeficiency virus type 1 (HIV-1) to CD4 receptors induces multiple cellular signaling pathways, including the MEK/ERK cascade. While the interaction of X4 HIV-1 with CXCR4 does not seem to activate this pathway, viruses using CCR5 for entry efficiently activate MEK/ERK kinases (W. Popik, J. E. Hesselgesser, and P. M. Pitha, J. Virol. 72:6406–6413, 1998; W. Popik and P. M. Pitha, Virology 252:210–217, 1998). Since the importance of MEK/ERK in the initial steps of viral replication is poorly understood, we have examined the role of MEK/ERK signaling in the CD3- and CD28 (CD3/CD28)-mediated activation of HIV-1 replication in resting peripheral blood CD4+T lymphocytes infected with X4 or R5 HIV-1. We have found that the MEK/ERK inhibitor U0126 selectively inhibited CD3/CD28-stimulated replication of X4 HIV-1, while it did not affect the replication of R5 HIV-1. Inhibition of the CD3/CD28-stimulated MEK/ERK pathway did not affect the formation of the early proviral transcripts in cells infected with either X4 or R5 HIV-1, indicating that virus reverse transcription is not affected in the absence of MEK/ERK signaling. In contrast, the levels of nuclear provirus in cells infected with X4 HIV-1, detected by the formation of circular proviral DNA, was significantly lower in cells stimulated in the presence of MEK/ERK inhibitor than in the absence of the inhibitor. However, in cells infected with R5 HIV-1, the inhibition of the MEK/ERK pathway did not affect nuclear localization of the proviral DNA. These data suggest that the nuclear import of X4, but not R5, HIV-1 is dependent on a CD3/CD28-stimulated MEK/ERK pathway.
Herpesvirus mRNAs Are Sorted for Export via Crm1-Dependent and -Independent Pathways
Authors: Soliman Tarik M. , Silverstein Saul J. 
Published: July 2002

ABSTRACT
Cellular pre-mRNA splicing is inhibited by ICP27, a herpes simplex virus regulatory protein, resulting in the shutoff of host protein synthesis. Here we reveal that ICP27 also mediates the export of some virus RNAs via a Crm1-dependent pathway and present evidence that independent domains are required for these functions. Sorting of some viral mRNAs for nuclear export requires Crm1, while other virus mRNAs are exported via another pathway.
Evolutionarily Related Sindbis-Like Plant Viruses Maintain Different Levels of Population Diversity in a Common Host
Authors: Schneider William L. , Roossinck Marilyn J. 
Published: July 2002

ABSTRACT

The levels of population diversity of three related Sindbis-like plant viruses,
Tobacco mosaic virus
(TMV),
Cucumber mosaic virus
(CMV), and
Cowpea chlorotic mottle virus
(CCMV), in infections of a common host,
Nicotiana benthamiana
, established from genetically identical viral RNA were examined. Despite probably having a common evolutionary ancestor, the three viruses maintained different levels of population diversity. CMV had the highest levels of diversity, TMV had an intermediate level of diversity, and CCMV had no measurable level of diversity in
N. benthamiana
. Interestingly, the levels of diversity were correlated to the relative host range sizes of the three viruses. The levels of diversity also remained relatively constant over the course of serial passage. Closer examination of the CMV and TMV populations revealed biases for particular types of substitutions and regions of the genome that may tolerate fewer mutations.
CD4+T-Cell-Mediated Antiviral Protection of the Upper Respiratory Tract in BALB/c Mice following Parenteral Immunization with a Recombinant Respiratory Syncytial Virus G Protein Fragment
Authors: Plotnicky-Gilquin Hélène , Robert Alain , Chevalet Laurent , Haeuw Jean-Francois , Beck Alain , Bonnefoy Jean-Yves , Brandt Christian , Siegrist Claire-Anne , Nguyen Thien Ngoc , Power Ultan F. 
Published: July 2002

ABSTRACTWe analyzed the protective mechanisms induced against respiratory syncytial virus subgroup A (RSV-A) infection in the lower and upper respiratory tracts (LRT and URT) of BALB/c mice after intraperitoneal immunization with a recombinant fusion protein incorporating residues 130 to 230 of RSV-A G protein (BBG2Na). Mother-to-offspring antibody (Ab) transfer and adoptive transfer of BBG2Na-primed B cells into SCID mice demonstrated that Abs are important for LRT protection but have no effect on URT infection. In contrast, RSV-A clearance in the URT was achieved in a dose-dependent fashion after adoptive transfer of BBG2Na-primed T cells, while it was abolished in BBG2Na-immunized mice upon in vivo depletion of CD4+, but not CD8+, T cells. Furthermore, the conserved RSV-A G protein cysteines and residues 193 and 194, overlapping the recently identified T helper cell epitope on the G protein (P. W. Tebbey et al., J. Exp. Med. 188:1967–1972, 1998), were found to be essential for URT but not LRT protection. Taken together, these results demonstrate for the first time that CD4+T cells induced upon parenteral immunization with an RSV G protein fragment play a critical role in URT protection of normal mice against RSV infection.
Molecular Characterization of a Bacteriophage (Chp2) from
Chlamydia psittaci
Authors: Liu B. L. , Everson J. S. , Fane B. , Giannikopoulou P. , Vretou E. , Lambden P. R. , Clarke I. N. 
Published: July 2002

ABSTRACT

Comparisons of the proteome of abortifacient
Chlamydia psittaci
isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine
C. psittaci
RBs. Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the φX174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein.
Polyomavirus-Infected Dendritic Cells Induce Antiviral CD8+T Lymphocytes
Authors: Drake Donald R. , Moser Janice M. , Hadley Annette , Altman John D. , Maliszewski Charles , Butz Eric , Lukacher Aron E. 
Published: July 2002

ABSTRACTCD8+T cells are critical for the clearance of acute polyomavirus infection and the prevention of polyomavirus-induced tumors, but the antigen-presenting cell(s) involved in generating polyomavirus-specific CD8+T cells have not been defined. We investigated whether dendritic cells and macrophages are permissive for polyomavirus infection and examined their potential for inducing antiviral CD8+T cells. Although dendritic cells and macrophages both supported productive polyomavirus infection, dendritic cells were markedly more efficient at presenting the immunodominant viral epitope to CD8+T cells. Additionally, infected dendritic cells, but not infected macrophages, primed anti-polyomavirus CD8+T cells in vivo. Treatment with Flt3 ligand, a hematopoietic growth factor that dramatically expands the number of dendritic cells, markedly enhanced the magnitude of virus-specific CD8+T-cell responses during acute infection and the pool of memory anti-polyomavirus CD8+T cells. These findings suggest that virus-infected dendritic cells induce polyomavirus-specific CD8+T cells in vivo and raise the potential for their use as cellular adjuvants to promote CD8+T cell surveillance against polyomavirus-induced tumors.
Consequences of Fas-Mediated Human Dendritic Cell Apoptosis Induced by Measles Virus
Authors: Servet-Delprat Christine , Vidalain Pierre-Olivier , Azocar Olga , Le Deist Françoise , Fischer Alain , Rabourdin-Combe Chantal 
Published: July 2002

ABSTRACTMortality from measles virus (MV) infection is caused mostly by secondary infections associated with a pronounced immunosuppression. Dendritic cells (DCs) represent a major target of MV and could be involved in immunosuppression. In this study, human monocyte-derived DCs were used to demonstrate that DC apoptosis in MV-infected DC–T-cell cocultures is Fas mediated, whereas apoptotic T cells could not be rescued by blocking the Fas pathway. Two novel consequences of DC apoptosis after MV infection were demonstrated. (i) Fas-mediated apoptosis of DCs facilitates MV release, while CD40 activation enhances MV replication in DCs. Indeed, detailed studies of infectious MV release and intracellular MV nucleoprotein (NP) showed that inhibition of CD40-CD40L ligand interaction blocks NP synthesis. We conclude that the CD40 ligand expressed by activated T cells first enhances MV replication in DCs, and then Fas ligand produced by activated T cells induces Fas-mediated apoptosis of DCs, thus facilitating MV release. (ii) Not only MV-infected DCs but also bystander uninfected DCs undergo a maturation process confirmed by CD1a, CD40, CD80, CD86, CD83, and major histocompatibility complex type II labeling. The bystander maturation effect results from contact and/or engulfment of MV-induced apoptotic DCs by uninfected DCs. A model is proposed to explain how both a specific immune response and immunosuppression can simultaneously occur after MV infection through Fas-mediated apoptosis and CD40 activation of DCs.
Vaccinia Virus Vectors with an Inactivated Gamma Interferon Receptor Homolog Gene (B8R) Are Attenuated In Vivo without a Concomitant Reduction in Immunogenicity
Authors: Verardi Paulo H. , Jones Leslie A. , Aziz Fatema H. , Ahmad Shabbir , Yilma Tilahun D. 
Published: July 2002

ABSTRACTThe vaccinia virus (VV) B8R gene encodes a secreted protein with homology to the gamma interferon (IFN-γ) receptor. In vitro, the B8R protein binds to and neutralizes the antiviral activity of several species of IFN-γ, including human and rat IFN-γ; it does not, however, bind significantly to murine IFN-γ. Here we report on the construction and characterization of recombinant VVs (rVVs) lacking the B8R gene. While the deletion of this gene had no effect on virus replication in vitro, rVVs lacking the B8R gene were attenuated for mice. There was a significant decrease in weight loss and mortality in normal mice, and nude mice survived significantly longer than did controls inoculated with parental virus. This is a surprising result considering the minimal binding of the B8R protein to murine IFN-γ and its failure to block the antiviral activity of this cytokine in vitro. Such reduction in virulence could not be determined in rats, since they are considerably more resistant to VV infection than are mice. Finally, deletion of the B8R gene had no detectable effects on humoral immune responses. Mice and rats vaccinated with the rVVs showed identical humoral responses to both homologous and heterologous genes expressed by VV. This study demonstrates that the deletion of the VV B8R gene leads to enhanced safety without a concomitant reduction in immunogenicity.
A Recombinant Newcastle Disease Virus with Low-Level V Protein Expression Is Immunogenic and Lacks Pathogenicity for Chicken Embryos
Authors: Mebatsion Teshome , Verstegen Stefan , De Vaan Leonarda T. C. , Römer-Oberdörfer Angela , Schrier Carla C. 
Published: July 2002

ABSTRACTNewcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3′-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5,000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.