bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reversal of Human Cytomegalovirus Major Immediate-Early Enhancer/Promoter Silencing in Quiescently Infected Cells via the Cyclic AMP Signaling Pathway
Authors: Keller Michael J. , Wu Allen W. , Andrews Janet I. , McGonagill Patrick W. , Tibesar Eric E. , Meier Jeffery L. 
Published: February 2007

ABSTRACT
The human cytomegalovirus (HCMV) major immediate-early (MIE) enhancer contains five functional cyclic AMP (cAMP) response elements (CRE). Because the CRE in their native context do not contribute appreciably to MIE enhancer/promoter activity in lytically infected human fibroblasts and NTera2 (NT2)-derived neurons, we postulated that they might have a role in MIE enhancer/promoter reactivation in quiescently infected cells. Here, we show that stimulation of the cAMP signaling pathway by treatment with forskolin (FSK), an adenylyl cyclase activator, greatly alleviates MIE enhancer/promoter silencing in quiescently infected NT2 neuronal precursors. The effect is immediate, independent of de novo protein synthesis, associated with the phosphorylation of ATF-1 serine 63 and CREB serine 133, dependent on protein kinase A (PKA) and the enhancer's CRE, and linked to viral-lytic-cycle advancement. Coupling of FSK treatment with the inhibition of either histone deacetylases or protein synthesis synergistically activates MIE gene expression in a manner suggesting that MIE enhancer/promoter silencing is optimally relieved by an interplay of multiple regulatory mechanisms. In contrast, MIE enhancer/promoter silence is not overcome by stimulation of the gamma interferon (IFN-γ) signaling pathway, despite the enhancer having two IFN-γ-activated-site-like elements. We conclude that stimulation of the cAMP/PKA signaling pathway drives CRE-dependent MIE enhancer/promoter activation in quiescently infected cells, thus exposing a potential mode of regulation in HCMV reactivation.
Guanidine Hydrochloride Inhibits Mammalian Orthoreovirus Growth by Reversibly Blocking the Synthesis of Double-Stranded RNA
Authors: Murray Kenneth E. , Nibert Max L. 
Published: February 2007

ABSTRACT

Millimolar concentrations of guanidine hydrochloride (GuHCl) are known to inhibit the replication of many plant and animal viruses having positive-sense RNA genomes. For example, GuHCl reversibly interacts with the nucleotide-binding region of poliovirus protein 2C
ATPase
, resulting in a specific inhibition of viral negative-sense RNA synthesis. The use of GuHCl thereby allows for the spatiotemporal separation of poliovirus gene expression and RNA replication and provides a powerful tool to synchronize the initiation of negative-sense RNA synthesis during in vitro replication reactions. In the present study, we examined the effect of GuHCl on mammalian orthoreovirus (MRV), a double-stranded RNA (dsRNA) virus from the family
Reoviridae
. MRV growth in murine L929 cells was reversibly inhibited by 15 mM GuHCl. Furthermore, 15 mM GuHCl provided specific inhibition of viral dsRNA synthesis while sparing both positive-sense RNA synthesis and viral mRNA translation. By using GuHCl to provide temporal separation of MRV gene expression and genome replication, we obtained evidence that MRV primary transcripts support sufficient protein synthesis to assemble morphologically normal viral factories containing functional replicase complexes. In addition, the coordinated use of GuHCl and cycloheximide allowed us to demonstrate that MRV dsRNA synthesis can occur in the absence of ongoing protein synthesis, although to only a limited extent. Future studies utilizing the reversible inhibition of MRV dsRNA synthesis will focus on elucidating the target of GuHCl, as well as the components of the MRV replicase complexes.
Stability of a Receptor-Binding Active Human Immunodeficiency Virus Type 1 Recombinant gp140 Trimer Conferred by Intermonomer Disulfide Bonding of the V3 Loop: Differential Effects of Protein Disulfide Isomerase on CD4 and Coreceptor Binding
Authors: Billington J. , Hickling T. P. , Munro G. H. , Halai C. , Chung R. , Dodson G. G. , Daniels R. S. 
Published: February 2007

ABSTRACT
Stable trimeric forms of human immunodeficiency virus recombinant gp140 (rgp140) are important templates for determining the structure of the glycoprotein to assist in our understanding of HIV infection and host immune response. Such information will aid the design of therapeutic drugs and vaccines. Here, we report the production of a highly stable and trimeric rgp140 derived from a HIV type 1 (HIV-1) subtype D isolate that may be suitable for structural studies. The rgp140 is functional in terms of binding to CD4 and three human monoclonal antibodies (17b, b12, and 2G12) that have broad neutralizing activities against a range of HIV-1 isolates from different subtypes. Treatment of rgp140 with protein disulfide isomerase (PDI) severely restricted 17b binding capabilities. The stable nature of the rgp140 was due to the lack of processing at the gp120/41 boundary and the presence of an intermonomer disulfide bond formed by the cysteines of the V3 loop. Further characterization showed the intermonomer disulfide bond to be a target for PDI processing. The relevance of these findings to the roles of the V3 domain and the timing of PDI action during the HIV infection process are discussed.
PrP
Sc
in Salivary Glands of Scrapie-Affected Sheep
Authors: Vascellari Marta , Nonno Romolo , Mutinelli Franco , Bigolaro Michela , Di Bari Michele Angelo , Melchiotti Erica , Marcon Stefano , D'Agostino Claudia , Vaccari Gabriele , Conte Michela , De Grossi Luigi , Rosone Francesca , Giordani Francesco , Agrimi Umberto 
Published: February 2007

ABSTRACT

The salivary glands of scrapie-affected sheep and healthy controls were investigated for the presence of the pathological prion protein (PrP
Sc
). PrP
Sc
was detected in major (parotid and mandibular) and minor (buccal, labial, and palatine) salivary glands of naturally and experimentally infected sheep. Using Western blotting, the PrP
Sc
concentration in glands was estimated to be 0.02 to 0.005% of that in brain. Immunohistochemistry revealed intracellular depositions of PrP
Sc
in ductal and acinar epithelia and occasional labeling in the lumina of salivary ducts. The presence of PrP
Sc
in salivary glands highlights the possible role of saliva in the horizontal transmission of scrapie.
Persistence of Cyprinid Herpesvirus 3 in Infected Cultured Carp Cells
Authors: Dishon Arnon , Davidovich Maya , Ilouze Maya , Kotler Moshe 
Published: February 2007

ABSTRACT
Cyprinid herpesvirus 3 (CyHV-3), previously designated carp interstitial nephritis and gill necrosis virus or koi herpesvirus, is the cause of a worldwide mortal disease of koi and carp. Morphologically, the virus resembles herpesviruses, yet it bears a genome of 277 to 295 kbp, which is divergent from most of the genomic sequences available in GenBank. The disease afflicts fish in the transient seasons, when the water temperature is 18 to 28°C, conditions which permit virus propagation in cultured cells. Here we report that infectious virus is preserved in cultured cells maintained for 30 days at 30°C. CyHV-3-infected vacuolated cells with deformed morphology converted to normal, and plaques disappeared following shifting up of the temperature and reappeared after transfer to the permissive temperature. Viral propagation and viral gene transcription were turned off by shifting cells to the nonpermissive temperature. Upon return of the cells to the permissive temperature, transcription of viral genes was reactivated in a sequence distinguished from that occurring in naïve cells following infection. Our results show that CyHV-3 persists in cultured cells maintained at the nonpermissive temperature and suggest that viruses could persist for long periods in the fish body, enabling a new burst of infection upon a shift to a permissive temperature.
Rab 5 Is Required for the Cellular Entry of Dengue and West Nile Viruses
Authors: Krishnan Manoj N. , Sukumaran Bindu , Pal Utpal , Agaisse Herve , Murray James L. , Hodge Thomas W. , Fikrig Erol 
Published: February 2007

ABSTRACT
The mechanisms of cellular entry of dengue and West Nile viruses are not well characterized. We show that both these viruses enter HeLa cells by clathrin-dependent endocytosis and require vacuolar acidic pH. Inhibition of the GTPase Rab 5 or 7, which regulates transport to early or late endosomes, respectively, demonstrated that Rab 5 was essential for survival of both dengue and West Nile virus. These data broaden our understanding of the pathways required for productive dengue and West Nile virus infection and may facilitate new strategies for combating disease.
Xenopus
Xp54 and Human RCK/p54 Helicases Functionally Replace Yeast Dhh1p in Brome Mosaic Virus RNA Replication
Authors: Alves-Rodrigues Isabel , Mas Antonio , Díez Juana 
Published: February 2007

ABSTRACT

By using a Brome mosaic virus (BMV)-
Saccharomyces cerevisiae
system, we previously showed that the cellular Lsm1p-7p/Pat1p/Dhh1p decapping-activator complex functions in BMV RNA translation and replication. As a first approach in investigating whether the corresponding human homologues play a similar role, we expressed human Lsm1p (hLsm1p) and RCK/p54 in yeast. Expression of RCK/p54 but not hLsm1p restored the defect in BMV RNA translation and replication observed in the
dhh1Δ
and
lsm1Δ
strains, respectively. This functional conservation, together with the common replication strategies of positive-stranded RNA viruses, suggests that RCK/p54 may also play a role in the replication of positive-stranded RNA viruses that infect humans.
Crystal Structure of the Dengue Virus RNA-Dependent RNA Polymerase Catalytic Domain at 1.85-Angstrom Resolution
Authors: Yap Thai Leong , Xu Ting , Chen Yen-Liang , Malet Helene , Egloff Marie-Pierre , Canard Bruno , Vasudevan Subhash G. , Lescar Julien 
Published: February 2007

ABSTRACT

Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the
Flavivirus
genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The
S
-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-Å resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.
Bax Is Activated during Rotavirus-Induced Apoptosis through the Mitochondrial Pathway
Authors: Martin-Latil Sandra , Mousson Laurence , Autret Arnaud , Colbère-Garapin Florence , Blondel Bruno 
Published: February 2007

ABSTRACT

Rotaviruses are the leading cause of infantile viral gastroenteritis worldwide. Mature enterocytes of the small intestine infected by rotavirus undergo apoptosis, and their replacement by less differentiated dividing cells probably leads to defective absorptive function of the intestinal epithelium, which, in turn, contributes to osmotic diarrhea and rotavirus pathogenesis. Here we show that infection of MA104 cells by the simian rhesus rotavirus strain RRV induced caspase-3 activation, DNA fragmentation, and cleavage of poly(ADP-ribose) polymerase; all three phenomena are features of apoptosis. RRV induced the release of cytochrome
c
from mitochondria to the cytosol, indicating that the mitochondrial apoptotic pathway was activated. RRV infection of MA104 cells activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change. Most importantly, Bax-specific small interfering RNAs partially inhibited cytochrome
c
release in RRV-infected cells. Thus, mitochondrial dysfunction induced by rotavirus is Bax dependent. Apoptosis presumably leads to impaired intestinal functions, so our findings contribute to improving our understanding of rotavirus pathogenesis at the cellular level.
Role of the CD8
+
Dendritic Cell Subset in Transmission of Prions
Authors: Sethi Shneh , Kerksiek Kristen M. , Brocker Thomas , Kretzschmar Hans 
Published: February 2007

ABSTRACT

Controversial results have been observed in mouse models regarding the role of lymphoid tissues in prion pathogenesis. To investigate the role of dendritic cells (DC), we used a transgenic mouse model. In this model (CD11c-N17Rac1), a significant reduction of CD8
+
CD11c
hi
DC has been described, and the remaining CD8
+
DC demonstrate a reduced capacity for the uptake of apoptotic cells. After intraperitoneal prion infection, significantly longer incubation times were observed in CD11c-N17Rac1 mice than in controls, indicating that a defect in CD8
+
CD11c
hi
DC significantly impedes neuroinvasion after intraperitoneal infection. In contrast, no distinct differences were observed between CD11c-N17Rac1 mice and controls after oral infection. This provides evidence that oral and intraperitoneal prion infections differ in lymphoreticular requirements.
Herpes Simplex Virus Infection Induces Phosphorylation and Delocalization of Emerin, a Key Inner Nuclear Membrane Protein
Authors: Morris James B. , Hofemeister Helmut , O'Hare Peter 
Published: February 2007

ABSTRACTThe inner nuclear membrane (INM) contains specialized membrane proteins that selectively interact with nuclear components including the lamina, chromatin, and DNA. Alterations in the organization of and interactions with INM and lamina components are likely to play important roles in herpesvirus replication and, in particular, exit from the nucleus. Emerin, a member of the LEM domain class of INM proteins, binds a number of nuclear components including lamins, the DNA-bridging protein BAF, and F-actin and is thought to be involved in maintaining nuclear integrity. Here we report that emerin is quantitatively modified during herpes simplex virus (HSV) infection. Modification begins early in infection, involves multiple steps, and is reversed by phosphatase treatment. Emerin phosphorylation during infection involves one or more cellular kinases but can also be influenced by the US3 viral kinase, a protein whose function is known to be involved in HSV nuclear egress. The results from biochemical extraction analyses and from immunofluorescence of the detergent-resistant population demonstrate that emerin association with the INM significantly reduced during infection. We propose that the induction of emerin phosphorylation in infected cells may be involved in nuclear egress and uncoupling interactions with targets such as the lamina, chromatin, or cytoskeletal components.
Adenovirus E4 ORF3 Protein Inhibits the Interferon-Mediated Antiviral Response
Authors: Ullman Amanda J. , Reich Nancy C. , Hearing Patrick 
Published: February 2007

ABSTRACT
The PML oncogenic domain (POD/ND10/PML body) is a common target of DNA viruses, which replicate their genomes in proximity to this nuclear structure. The adenovirus early protein E4 ORF3 is both necessary and sufficient to rearrange PODs from punctate bodies into track-like structures. Although multiple hypotheses exist, the precise reason for this activity has not yet been elucidated. PML, the protein responsible for nucleating PODs, is an interferon (IFN)-stimulated gene, implicating the participation of this nuclear body in an innate antiviral response. Here, we demonstrate that E4 ORF3 is critical to the replicative success of adenovirus during the IFN-induced antiviral state. When cells are pretreated with either IFN-α or IFN-γ, a mutant virus that does not express E4 ORF3 is severely compromised for replication. This result suggests the functional significance of ORF3 track formation is the inhibition of a POD-mediated, antiviral mechanism. Replication of the E4 ORF3 mutant virus can be rescued following the introduction of E4 ORF3 from evolutionarily divergent adenoviruses, suggesting a conserved function for E4 ORF3 inhibition of the IFN-induced antiviral state. Furthermore, E4 ORF3 inhibition of an IFN-induced response is unrelated to the inhibition of adenovirus replication by the Mre11-Rad50-Nbs1 DNA repair complex. We propose that the evolutionarily conserved function of the adenovirus E4 ORF3 protein is the inhibition of a host interferon response to viral infection via disruption of the PML oncogenic domain.
Distinct RNA Elements Confer Specificity to Flavivirus RNA Cap Methylation Events
Authors: Dong Hongping , Ray Debashish , Ren Suping , Zhang Bo , Puig-Basagoiti Francesc , Takagi Yuko , Ho C. Kiong , Li Hongmin , Shi Pei-Yong 
Published: February 2007

ABSTRACT

The 5′ end of the flavivirus plus-sense RNA genome contains a type 1 cap (m
7
GpppAmG), followed by a conserved stem-loop structure. We report that nonstructural protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5′ terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2′-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5′ stem-loop structure; in contrast, 2′-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5′ viral RNA of 20 nucleotides. The cap analogues GpppA and m
7
GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m
7
Gppp-terminated RNAs suggest that the 5′ termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery.
Induction of Mutations in
Drosophila melanogaster gypsy
Retroelements by Modulation of Intracellular Deoxynucleoside Triphosphate Pools In Vivo
Authors: Terzian Christophe , Henry Michel , Meyerhans Andreas , Wain-Hobson Simon , Vartanian Jean-Pierre 
Published: February 2007

ABSTRACT

The retroviral mutation rate is susceptible to a number of variables, including the balance between intracellular deoxynucleoside triphosphate (dNTP) pools. While this follows from tissue culture studies, the issue has never been addressed directly in vivo. To explore this question in a tractable experimental system, we analyzed the impact of thymidine treatment on the synthesis of
gypsy
retroelement cDNA from
Drosophila melanogaster
during development through to hatching. The mutation frequency was enhanced approximately 16-fold over the levels seen in the experimental background. Due to the lack of proofreading, these
gypsy
elements represent hypervariable loci within the
Drosophila
genome, suggesting that dNTP pool imbalances in vivo are mutagenic.
Vesicular Stomatitis Viruses Resistant to the Methylase Inhibitor Sinefungin Upregulate RNA Synthesis and Reveal Mutations That Affect mRNA Cap Methylation
Authors: Li Jianrong , Chorba John S. , Whelan Sean P. J. 
Published: February 2007

ABSTRACT

Sinefungin (SIN), a natural
S
-adenosyl-
l
-methionine analog produced by
Streptomyces griseolus
, is a potent inhibitor of methyltransferases. We evaluated the effect of SIN on replication of vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses. The 241-kDa large polymerase (L) protein of VSV methylates viral mRNA cap structures at the guanine-N-7 (G-N-7) and ribose-2′-O (2′-O) positions. By performing transcription reactions in vitro, we show that both methylations are inhibited by SIN and that methylation was more sensitive at the G-N-7 than at 2′-O position. We further show that SIN inhibited growth of VSV in cell culture, reducing viral yield by 50-fold and diminishing plaque size. We isolated eight mutants that were resistant to SIN as judged by their growth characteristics. The SIN-resistant (SIN
R
) viruses contained mutations in the
L
gene, the promoter for
L
gene expression provided by the conserved sequence elements of the
G
-
L
gene junction and the
M
gene. Five mutations resulted in amino acid substitutions to conserved regions II/III and VI of the L protein. For each mutant, we examined viral gene expression in cells and cap methylation in vitro. SIN
R
mutants upregulated RNA synthesis in the presence of SIN, which may be responsible for their resistance. We also found that some SIN
R
viruses with
L
gene mutations were defective in cap methylation in vitro, yet their methylases were less sensitive to SIN inhibition than those of the wild-type parent. These studies show that the VSV methylases are inhibited by SIN, and they define new regions of L protein that affect cap methylation. These studies also provide experimental evidence that inhibition of cap methylases is a potential strategy for development of antiviral therapeutics against nonsegmented negative-strand RNA viruses.
Switching the Substrate Specificity of the Two-Component NS2B-NS3 Flavivirus Proteinase by Structure-Based Mutagenesis
Authors: Shiryaev Sergey A. , Ratnikov Boris I. , Aleshin Alexander E. , Kozlov Igor A. , Nelson Nicholas A. , Lebl Michal , Smith Jeffrey W. , Liddington Robert C. , Strongin Alex Y. 
Published: February 2007

ABSTRACT

The flavivirus NS2B-NS3(pro)teinase is an essential element in the proteolytic processing of the viral precursor polyprotein and therefore a potential drug target. Recently, crystal structures and substrate preferences of NS2B-NS3pro from Dengue and West Nile viruses (DV and WNV) were determined. We established that the presence of Gly-Gly at the P1′-P2′ positions is optimal for cleavage by WNV NS3pro, whereas DV NS3pro tolerates well the presence of bulky residues at either P1′ or P2′. Structure-based modeling suggests that Arg
76
and Pro
131
-Thr
132
limit the P1′-P2′ subsites and restrict the cleavage preferences of the WNV enzyme. In turn, Leu
76
and Lys
131
-Pro
132
widen the specificity of DV NS3pro. Guided by these structural models, we expressed and purified mutant WNV NS2B-NS3pro and evaluated cleavage preferences by using positional scanning of the substrate peptides in which the P4-P1 and the P3′-P4′ positions were fixed and the P1′ and P2′ positions were each randomized. We established that WNV R76L and P131K-T132P mutants acquired DV-like cleavage preferences, whereas T52V had no significant effect. Our work is the first instance of engineering a viral proteinase with switched cleavage preferences and should provide valuable data for the design of optimized substrates and substrate-based selective inhibitors of flaviviral proteinases.