bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genotyping of Porcine Endogenous Retroviruses from a Family of Miniature Swine
Authors: Quinn Gary , Wood James , Suling Kristen , Arn Scott , Sachs David H. , Schuurman Henk-Jan , Patience Clive 
Published: December 2003

ABSTRACT

The identification of animals in an inbred miniature swine herd that consistently fail to produce replication- competent humantropic porcine endogenous retrovirus (PERV) has prompted studies on the biology of PERV in transmitter and nontransmitter animals. We analyzed PERV RNA transcript profiles in a family of inbred miniature swine (SLA
d/d
haplotype) in which individual members differed in their capacity to generate humantropic and ecotropic (i.e., pigtropic) virus. We identified unique
Hae
III and
Hpa
II
gag
restriction fragment length polymorphism (RFLP) profiles resulting from single nucleotide polymorphisms in blood cells; these were found only in animals that produced humantropic PERV. These
Hae
III and
Hpa
II
gag
RFLP profiles proved to be components of humantropic PERV as they were transmitted to 293 human target cells in vitro. The humantropic
Hae
III and
Hpa
II
gag
RFLP genotypes in the family of study were not present in other miniature swine in the herd that produced humantropic PERV, indicating that these RFLP profiles relate specifically to this family's lineage.
Cholesterol Removal by Methyl-β-Cyclodextrin Inhibits Poliovirus Entry
Authors: Danthi Pranav , Chow Marie 
Published: December 2003

ABSTRACT
Upon binding to the poliovirus receptor (PVR), the poliovirus 160S particles undergo a conformational transition to generate 135S particles, which are believed to be intermediates in the virus entry process. The 135S particles interact with host cell membranes through exposure of the N termini of VP1 and the myristylated VP4 protein, and successful cytoplasmic delivery of the genomic RNA requires the interaction of these domains with cellular membranes whose identity is unknown. Because detergent-insoluble microdomains (DIMs) in the plasma membrane have been shown to be important in the entry of other picornaviruses, it was of interest to determine if poliovirus similarly required DIMs during virus entry. We show here that methyl-β-cyclodextrin (MβCD), which disrupts DIMs by depleting cells of cholesterol, inhibits virus infection and that this inhibition was partially reversed by partially restoring cholesterol levels in cells, suggesting that MβCD inhibition of virus infection was mediated by removal of cellular cholesterol. However, fractionation of cellular membranes into DIMs and detergent-soluble membrane fractions showed that both PVR and poliovirus capsid proteins localize not to DIMs but to detergent-soluble membrane fractions during entry into the cells, and their localization was unaffected by treatment with MβCD. We further demonstrate that treatment with MβCD inhibits RNA delivery after formation of the 135S particles. These data indicate that the cholesterol status of the cell is important during the process of genome delivery and that these entry pathways are distinct from those requiring DIM integrity.
The Genome of Canarypox Virus
Authors: Tulman E. R. , Afonso C. L. , Lu Z. , Zsak L. , Kutish G. F. , Rock D. L. 
Published: December 2003

ABSTRACT
Here we present the genomic sequence, with analysis, of a canarypox virus (CNPV). The 365-kbp CNPV genome contains 328 potential genes in a central region and in 6.5-kbp inverted terminal repeats. Comparison with the previously characterized fowlpox virus (FWPV) genome revealed avipoxvirus-specific genomic features, including large genomic rearrangements relative to other chordopoxviruses and novel cellular homologues and gene families. CNPV also contains many genomic differences with FWPV, including over 75 kbp of additional sequence, 39 genes lacking FWPV homologues, and an average of 47% amino acid divergence between homologues. Differences occur primarily in terminal and, notably, localized internal genomic regions and suggest significant genomic diversity among avipoxviruses. Divergent regions contain gene families, which overall comprise over 49% of the CNPV genome and include genes encoding 51 proteins containing ankyrin repeats, 26 N1R/p28-like proteins, and potential immunomodulatory proteins, including those similar to transforming growth factor β and β-nerve growth factor. CNPV genes lacking homologues in FWPV encode proteins similar to ubiquitin, interleukin-10-like proteins, tumor necrosis factor receptor, PIR1 RNA phosphatase, thioredoxin binding protein, MyD116 domain proteins, circovirus Rep proteins, and the nucleotide metabolism proteins thymidylate kinase and ribonucleotide reductase small subunit. These data reveal genomic differences likely affecting differences in avipoxvirus virulence and host range, and they will likely be useful for the design of improved vaccine vectors.
EBNA3C Coactivation with EBNA2 Requires a SUMO Homology Domain
Authors: Rosendorff Adam , Illanes Diego , David Gregory , Lin Jeffrey , Kieff Elliott , Johannsen Eric 
Published: December 2003

ABSTRACT

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is critical for EBV immortalization of infected B lymphocytes and can coactivate the EBV LMP1 promoter with EBNA2. EBNA3C amino acids 365 to 545 are necessary and sufficient for coactivation and are required for SUMO-1 and SUMO-3 interaction. We found that EBNA3C but not EBNA3CΔ343-545 colocalized with SUMO-1 in nuclear bodies and was modified by SUMO-2, SUMO-3, and SUMO-1. EBNA3C amino acids 545 to 628 and amino acids 30 to 365 were also required for EBNA3C sumolation and nuclear body localization but were dispensable for coactivation, indicating that EBNA3C sumolation is not required for coactivation. Furthermore, EBNA3C amino acids 476 to 992 potently coactivated with EBNA2 but EBNA3C amino acids 516 to 922 lacked activity, indicating that amino acids 476 to 515 are critical for coactivation. EBNA3C amino acids 476 to 515 include DDDVIEV
507-513
, which are similar to SUMO-1 EEDVIEV
84-90
. EBNA3C m1 and m2 point mutations, DDD
507-509
mutated to AAA and DVIEVID
509-513
mutated to AVIAVIA, respectively, diminished SUMO-1 and SUMO-3 interaction in directed yeast two-hybrid and glutathione
S
-transferase pulldown assays. Furthermore, EBNA3C m1 and m2 did not coactivate the LMP1 promoter with EBNA2. Overexpression of wild-type SUMO-1, SUMO-3, and the SUMO-conjugating enzyme UBC9 coactivated the LMP1 promoter with EBNA2. Since EBNA2 activation is dependent on p300/CBP, the possible effect of EBNA3C on p300-mediated transcription was assayed. EBNA3C potentiated transcription of p300 fused to a heterologous DNA binding domain, whereas EBNA3C m1 and m2 did not. All of these data are consistent with a model in which EBNA3C upregulates EBNA2-mediated gene activation by binding to a sumolated repressor and inhibiting repressive effects on p300/CBP and other transcription factor(s) at EBNA2-regulated promoters.
An External Loop Region of Domain III of Dengue Virus Type 2 Envelope Protein Is Involved in Serotype-Specific Binding to Mosquito but Not Mammalian Cells
Authors: Hung Jan-Jong , Hsieh Meng-Ti , Young Ming-Jer , Kao Chuan-Liang , King Chwan-Chuen , Chang Wen 
Published: December 2003

ABSTRACT
Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.
Herpes Simplex Virus Type 1 Primary Envelopment: UL34 Protein Modification and the US3-UL34 Catalytic Relationship
Authors: Ryckman Brent J. , Roller Richard J. 
Published: December 2003

ABSTRACT
The herpes simplex virus type 1 (HSV-1) US3 kinase is likely important for primary envelopment of progeny nucleocapsids since it localizes to the nuclear envelope of infected cells and largely determines the phosphorylation state and localization of the necessary primary envelopment factor, the UL34 protein. In HEp-2 cells, the production of infectious US3 null progeny is delayed and decreased relative to that of the parental strain, HSV-1(F). Furthermore, the US3 kinase affects the morphology of primary envelopment such that in its absence, UL34 protein-containing enveloped virions accumulate within membrane-bound vesicles. These vesicles are most often found along the interior periphery of the nucleus and may be derived from the inner nuclear membrane. Since the US3 and UL34 proteins comprise a kinase-substrate pair, a reasonable hypothesis is that the US3 kinase influences these replication parameters by direct phosphorylation of the UL34 protein. For this report, recombinant viruses were constructed to determine the significance of UL34 protein phosphorylation and US3 catalytic activity on UL34 protein localization, single-step growth, and envelopment morphology in both HEp-2 and Vero cells. The data presented suggest that the significance of UL34 phosphorylation is cell type dependent and that efficient viral morphogenesis requires US3-mediated phosphorylation of an infected cell protein other than UL34.
Structural and Functional Analysis of Human Cytomegalovirus US3 Protein
Authors: Misaghi Shahram , Sun Zhen-Yu J. , Stern Patrick , Gaudet Rachelle , Wagner Gerhard , Ploegh Hidde 
Published: December 2003

ABSTRACTHuman cytomegalovirus (HCMV) unique short region 3 (US3) protein, a type I membrane protein, prevents maturation of class I major histocompatibility complex (MHC) molecules by retaining them in the endoplasmic reticulum (ER) and thus helps inhibit antigen presentation to cytotoxic T cells. US3 molecules bind to class I MHC molecules in a transient fashion but retain them very efficiently in the ER nonetheless. The US3 luminal domain is responsible for ER retention of US3 itself, while both the US3 luminal and transmembrane domains are necessary for retaining class I MHC in the ER. We have expressed the luminal domain of US3 molecule inEscherichia coliand analyzed its secondary structure by using nuclear magnetic resonance. We then predicted the US3 tertiary structure by modeling it based on the US2 structure. Unlike the luminal domain of US2, the US3 luminal domain does not obviously interact with class I MHC molecules. The luminal domain of US3 dynamically oligomerizes in vitro and full-length US3 molecules associate with each other in vivo. We present a model depicting how dynamic oligomerization of US3 may enhance its ability to retain class I molecules within the ER.
Identification and Characterization of Novel, Naturally Processed Measles Virus Class II HLA-DRB1 Peptides
Authors: Ovsyannikova Inna G. , Johnson Kenneth L. , Muddiman David C. , Vierkant Robert A. , Poland Gregory A. 
Published: December 2003

ABSTRACTPreviously, we identified a naturally processed and presented measles virus (MV) 19-amino-acid peptide, ASDVETAEGGEIHELLRLQ (MV-P), derived from the phosphoprotein and eluted from the human leukocyte antigen (HLA) class II molecule by using mass spectrometry. We report here the identification of a 14-amino-acid peptide, SAGKVSSTLASELG, derived from the MV nucleoprotein (MV-N) bound to HLA-DRB1*0301. Peripheral blood mononuclear cells (PBMC) from 281 previously vaccinated measles-mumps-rubella II (MMR-II) subjects (HLA discordant) were studied for peptide recognition by T cells. Significant gamma interferon (IFN-γ) responses to MV-P and MV-N peptides were observed in 55.9 and 15.3% of subjects, respectively. MV-P- and MV-N-specific interleukin-4 (IL-4) responses were detected in 19.2 and 23.1%, respectively, of PBMC samples. Peptide-specific cytokine responses and HLA-DRB1 allele associations revealed that, for the MV-P peptide, the allele with the strongest association with both IFN-γ (P= 0.02) and IL-4 (P= 0.03) secretion was DRB1*0301. For MV-N, the allele with the strongest association with IFN-γ secretion was DRB1*1501 (P= 0.04), and the alleles with the strongest associations with IL-4 secretion were DRB1*1103 and DRB1*1303 (P= 0.01). These results indicate that HLA class II MV proteins can be processed, presented, and identified, and the ability to generate cell-mediated immune responses can be demonstrated. This information is promising for new vaccine design strategies with peptide-based vaccines.
The Rep Protein of Adeno-Associated Virus Type 2 Interacts with Single-Stranded DNA-Binding Proteins That Enhance Viral Replication
Authors: Stracker Travis H. , Cassell Geoffrey D. , Ward Peter , Loo Yueh-Ming , van Breukelen Bas , Carrington-Lawrence Stacy D. , Hamatake Robert K. , van der Vliet Peter C. , Weller Sandra K. , Melendy Thomas , Weitzman Matthew D. 
Published: December 2003

ABSTRACT
Adeno-associated virus (AAV) type 2 is a human parvovirus whose replication is dependent upon cellular proteins as well as functions supplied by helper viruses. The minimal herpes simplex virus type 1 (HSV-1) proteins that support AAV replication in cell culture are the helicase-primase complex of UL5, UL8, and UL52, together with the UL29 gene product ICP8. We show that AAV and HSV-1 replication proteins colocalize at discrete intranuclear sites. Transfections with mutant genes demonstrate that enzymatic functions of the helicase-primase are not essential. The ICP8 protein alone enhances AAV replication in an in vitro assay. We also show localization of the cellular replication protein A (RPA) at AAV centers under a variety of conditions that support replication. In vitro assays demonstrate that the AAV Rep68 and Rep78 proteins interact with the single-stranded DNA-binding proteins (ssDBPs) of Ad (Ad-DBP), HSV-1 (ICP8), and the cell (RPA) and that these proteins enhance binding and nicking of Rep proteins at the origin. These results highlight the importance of intranuclear localization and suggest that Rep interaction with multiple ssDBPs allows AAV to replicate under a diverse set of conditions.
Cultured Peripheral Neuroglial Cells Are Highly Permissive to Sheep Prion Infection
Authors: Archer Fabienne , Bachelin Corinne , Andreoletti Olivier , Besnard Nathalie , Perrot Gregory , Langevin Christelle , Le Dur Annick , Vilette Didier , Baron-Van Evercooren Anne , Vilotte Jean-Luc , Laude Hubert 
Published: December 2003

ABSTRACTTransmissible spongiform encephalopathies arise as a consequence of infection of the central nervous system (CNS) by prions. Spreading of the infectious agent through the peripheral nervous system (PNS) may represent a crucial step toward CNS neuroinvasion, but the modalities of this process have yet to be clarified. Here we provide further evidence that PNS glial cells are likely targets for infection by prions. Glial cell clones originating from dorsal root ganglia of transgenic mice expressing ovine PrP (tgOv) and simian virus 40 T antigen were found to be readily infectible by sheep scrapie agent. This led us to establish two stable cell lines that exhibited features of Schwann cells. These cells were shown to sustain an efficient and stable replication of sheep prion based on the high level of accumulation of abnormal PrP and infectivity in exposed cultures. We also provide evidence for abnormal PrP deposition in peripheral neuroglial cells from scrapie-infected tgOv mice and sheep. These findings have potential implications in terms of designing new cell systems permissive to prions and of peripheral pathobiology of prion infections.
Chromosome-Protein Interactions in Polyomavirus Virions
Authors: Carbone Mariarosaria , Ascione Giuseppe , Chichiarelli Silvia , Garcia Marie-Isabelle , Eufemi Margherita , Amati Paolo 
Published: December 2003

ABSTRACT

In this work, we sought to determine whether the components of the murine polyomavirus capsid establish specific interactions with the minichromosome encapsidated into the mature viral particles by using the
cis
-diamminedichloroplatinum(II) cross-linking reagent. Our data indicated that VP1, but not minor capsid proteins, interacts with the viral genome in vivo. In addition, semiquantitative PCR assays performed on cross-linked DNA complexes revealed that VP1 binds to all regions of the viral genome but significantly more to the regulatory region. The implications of such an interaction for viral infectivity are discussed.
Failure of Thymidine Kinase-Negative Herpes Simplex Virus To Reactivate from Latency following Efficient Establishment
Authors: Chen Shih-Heng , Pearson Angela , Coen Donald M. , Chen Shun-Hua 
Published: December 2003

ABSTRACT
Thymidine kinase-negative mutants of herpes simplex virus did not reactivate from latency in mouse trigeminal ganglia, even when their latent viral loads were comparable to those that permitted reactivation by wild-type virus. Thus, reduced establishment of latency does not suffice to account for the failure to reactivate.
Tetracycline-Inducible Packaging Cell Line for Production of Flavivirus Replicon Particles
Authors: Harvey Tracey J. , Liu Wen Jun , Wang Xiang Ju , Linedale Richard , Jacobs Michael , Davidson Andrew , Le Thuy T. T. , Anraku Itaru , Suhrbier Andreas , Shi Pei-Yong , Khromykh Alexander A. 
Published: December 2003

ABSTRACT

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 × 10
9
VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 10
10
VLPs per 10
6
transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8
+
-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 × 10
7
VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10
6
splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.
Rapid Genetic Engineering of Human Cytomegalovirus by Using a Lambda Phage Linear Recombination System: Demonstration that pp28 (UL99) Is Essential for Production of Infectious Virus
Authors: Britt William J. , Jarvis Michael , Seo Jun-Young , Drummond Derek , Nelson Jay 
Published: December 2003

ABSTRACT

A highly efficient lambda phage recombination system previously utilized for studies of bacterial artificial chromosome (BAC)-maintained mouse chromosomal DNA was adapted for the study of the role of human cytomegalovirus (HCMV)-encoded pp28 (UL99) in virus replication. Incorporating a two-step mutagenesis strategy with blue/white selection in
Escherichia coli
containing a HCMV AD169 BAC, we have shown that we can rapidly introduce point mutations into the HCMV BAC using linear PCR fragments. All manipulations were carried out in bacteria, which greatly accelerated the introduction and analysis of mutations in the viral genome. Our results indicated that HCMV pp28 was essential for the production of infectious virus and that introduction of a single base change that resulted in loss of the myristylation site on pp28 was also associated with the lack of production of infectious virus. Although the block in the viral morphogenesis cannot be determined from these studies, the latter finding suggested that authentic intracellular localization of pp28, not only the expression of the protein, is required for virus assembly.
Epstein-Barr Virus Immediate-Early Protein BZLF1 Inhibits Tumor Necrosis Factor Alpha-Induced Signaling and Apoptosis by Downregulating Tumor Necrosis Factor Receptor 1
Authors: Morrison Thomas E. , Mauser Amy , Klingelhutz Aloysius , Kenney Shannon C. 
Published: December 2003

ABSTRACTTumor necrosis factor alpha (TNF-α) is a key mediator of host immune and inflammatory responses and inhibits herpesvirus replication by cytolytic and noncytolytic mechanisms. TNF-α effects are primarily mediated through the major TNF-α receptor, TNF-R1, which is constitutively expressed in most cell types. Here we show that the Epstein-Barr virus (EBV) immediate-early protein BZLF1 prevents TNF-α activation of target genes and TNF-α-induced cell death. These effects are mediated by down-regulation of the promoter forTNF-R1.Additionally, we demonstrate that expression of TNF-R1 is downregulated during the EBV lytic replication cycle. Thus, EBV has developed a novel mechanism for evading TNF-α antiviral effects during lytic reactivation or primary infection.