bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Severe Acute Respiratory Syndrome Coronavirus Replicase Products and Characterization of Papain-Like Protease Activity
Authors: Harcourt Brian H. , Jukneliene Dalia , Kanjanahaluethai Amornrat , Bechill John , Severson Kari M. , Smith Catherine M. , Rota Paul A. , Baker Susan C. 
Published: November 2004

ABSTRACT

Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe
trans
-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione
S
-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a ≈300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro
trans
-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in
trans
at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.
Dissection of the Kaposi's Sarcoma-Associated Herpesvirus Gene Expression Program by Using the Viral DNA Replication Inhibitor Cidofovir
Authors: Lu Michael , Suen Jacqueline , Frias Carolina , Pfeiffer Ruth , Tsai Mong-Hsun , Chuang Eric , Zeichner Steven L. 
Published: November 2004

ABSTRACT
Treatment of primary effusion lymphoma cells latently infected by Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus-8 [HHV-8]) with agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a lytic viral replication cycle, with an ordered gene expression program. Initial studies of the KSHV expression program following TPA induction using viral microarrays yielded useful information concerning the viral expression program, but precise kinetic assignments for some genes remained unclear. Classically, late herpesvirus genes require viral DNA replication for maximal expression. We used cidofovir (CDV), a nucleotide-analogue KSHV DNA polymerase inhibitor, to dissect KSHV expression into two components: genes expressed without viral DNA replication and those requiring it. The expression of known immediate-early or early genes (e.g., open reading frames [ORFs] 50, K8 bZIP, and 57) serving lytic regulatory roles was relatively unaffected by the presence of CDV, while known late capsid and tegument structural genes (e.g., ORFs 25, 26, 64, and 67) were CDV sensitive. Latency-associated transcript ORF 73 was unaffected by the presence of TPA or CDV, suggesting that it was constitutively expressed. Expression of several viral cellular gene homologs, including K2 (vIL-6), ORF 72 (vCyclin), ORF 74 (vGPCR), and K9 (vIRF-1), was unaffected by the presence of CDV, while that of others, such as K4.1 (vMIP-III), K11.1 (vIRF-2), and K10.5 (LANA2, vIRF-3), was inhibited. The results distinguish KSHV genes whose full expression required viral DNA replication from those that did not require it, providing additional insights into KSHV replication and pathogenesis strategies and helping to show which viral cell homologs are expressed at particular times during the lytic process.
Accelerated Prion Disease in the Absence of Interleukin-10
Authors: Thackray Alana M. , McKenzie Andrew N. , Klein Michael A. , Lauder Angus , Bujdoso Raymond 
Published: November 2004

ABSTRACTThe identity of pro- and anti-inflammatory cytokines in the neuropathogenesis of prion diseases remains undefined. Here we have investigated the role of anti-inflammatory cytokines on the progression of prion disease through the use of mice that lack interleukin-4 (IL-4), IL-10, IL-13, or both IL-4 and IL-13. Collectively our data show that among these anti-inflammatory cytokines, IL-10 plays a prominent role in the regulation of prion disease. Mice deficient in IL-10 are highly susceptible to the development of prion disease and show a markedly shortened incubation time. In addition, we have correlated cytokine gene expression in prion-inoculated IL-10−/−mice to wild-type-inoculated animals. Our experiments show that in the absence of IL-10 there is an early expression of tumor necrosis factor alpha (TNF-α). In wild-type prion-inoculated mice, the expression of TNF-α mRNA occurs at a later time point that correlates with the extended incubation time for terminal disease development in these animals compared to those that lack IL-10. Elevated levels of IL-13 mRNA are found at early time points in the central nervous system of prion-inoculated IL-10−/−mice. At terminal disease, the brains of wild-type mice inoculated with RML or ME7 are characterized by elevated levels of mRNA for the proinflammatory cytokines TNF-α and IL-1β, together with the anti-inflammatory cytokines IL-10, IL-13, and transforming growth factor beta. Our data are consistent with a role for proinflammatory cytokines in the initiation of pathology during prion disease and an attempt by anti-inflammatory cytokines to regulate the ensuing, invariably fatal pathology.
Genome Assembly and Particle Maturation of the Birnavirus Infectious Pancreatic Necrosis Virus
Authors: Villanueva Rodrigo A. , Galaz José L. , Valdés Juan A. , Jashés Matilde M. , Sandino Ana María 
Published: November 2004

ABSTRACTIn this study, we have analyzed the morphogenesis of the birnavirus infectious pancreatic necrosis virus throughout the infective cycle in CHSE-214 cells by using a native agarose electrophoresis system. Two types of viral particles (designated A and B) were identified, isolated, and characterized both molecularly and biologically. Together, our results are consistent with a model of morphogenesis in which the genomic double-stranded RNA is immediately assembled, after synthesis, into a large (66-nm diameter) and uninfectious particle A, where the capsid is composed of both mature and immature viral polypeptides. Upon maturation, particles A yield particles B through the proteolytic cleavage of most of the remaining viral precursors within the capsid, the compaction of the particle (60-nm diameter), and the acquisition of infectivity. These studies will provide the foundation for further analyses of birnavirus particle assembly and RNA replication.
Overlapping CRE and E Box Motifs in the Enhancer Sequences of the Bovine Leukemia Virus 5′ Long Terminal Repeat Are Critical for Basal and Acetylation-Dependent Transcriptional Activity of the Viral Promoter: Implications for Viral Latency
Authors: Calomme Claire , Dekoninck Ann , Nizet Séverine , Adam Emmanuelle , Nguyên Thi Liên-Anh , Van Den Broeke Anne , Willems Luc , Kettmann Richard , Burny Arsène , Van Lint Carine 
Published: November 2004

ABSTRACT

Bovine leukemia virus (BLV) infection is characterized by viral latency in a large proportion of cells containing an integrated provirus. In this study, we postulated that mechanisms directing the recruitment of deacetylases to the BLV 5′ long terminal repeat (LTR) could explain the transcriptional repression of viral expression in vivo. Accordingly, we showed that BLV promoter activity was induced by several deacetylase inhibitors (such as trichostatin A [TSA]) in the context of episomal LTR constructs and in the context of an integrated BLV provirus. Moreover, treatment of BLV-infected cells with TSA increased H4 acetylation at the viral promoter, showing a close correlation between the level of histone acetylation and transcriptional activation of the BLV LTR. Among the known
cis
-regulatory DNA elements located in the 5′ LTR, three E box motifs overlapping cyclic AMP responsive elements (CREs) in U3 were shown to be involved in transcriptional repression of BLV basal gene expression. Importantly, the combined mutations of these three E box motifs markedly reduced the inducibility of the BLV promoter by TSA. E boxes are susceptible to recognition by transcriptional repressors such as Max-Mad-mSin3 complexes that repress transcription by recruiting deacetylases. However, our in vitro binding studies failed to reveal the presence of Mad-Max proteins in the BLV LTR E box-specific complexes. Remarkably, TSA increased the occupancy of the CREs by CREB/ATF. Therefore, we postulated that the E box-specific complexes exerted their negative cooperative effect on BLV transcription by steric hindrance with the activators CREB/ATF and/or their transcriptional coactivators possessing acetyltransferase activities. Our results thus suggest that the overlapping CRE and E box elements in the BLV LTR were selected during evolution as a novel strategy for BLV to allow better silencing of viral transcription and to escape from the host immune response.
Role of Nedd4 and Ubiquitination of Rous Sarcoma Virus Gag in Budding of Virus-Like Particles from Cells
Authors: Vana Marcy L. , Tang Yi , Chen Aiping , Medina Gisselle , Carter Carol , Leis Jonathan 
Published: November 2004

ABSTRACT

Rous sarcoma virus (RSV) budding requires an interaction of the L domain within the p2b region of Gag with cellular Nedd4-family E3 ubiquitin protein ligases. Members of our laboratories previously demonstrated that overexpression of a fragment of the chicken Nedd4-like protein (LDI-1 WW) inhibits Gag release in a dominant-negative manner (A. Kikonyogo, F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis, Proc. Natl. Acad. Sci. USA
98:
11199-11204, 2001). We have now identified the complete 3′ end of LDI-1 and determined that it has a C-terminal ubiquitin ligase HECT domain, similar to other Nedd4 family members. While overexpression of the full-length LDI-1 clone (LDI-1 FL) had little effect on Gag budding, an LDI-1 FL mutant with a substitution in the HECT domain catalytic site blocked Gag release, similar to LDI-1 WW. The coexpression of Gag and hemagglutinin-tagged ubiquitin (HA-Ub) resulted in the detection of mono- and polyubiquitinated forms of Gag in cells and mostly monoubiquitinated Gag in virus-like particles (VLPs). When the Nedd4-binding site (L domain) was deleted, ubiquitinated Gag was not detected. Interestingly, the release of Gag with ubiquitin covalently linked to the C terminus (Gag-Ub) was still blocked by LDI-1 WW. To understand the mechanism of this inhibition, we examined cells expressing Gag and LDI-1 WW by electron microscopy. In the presence of LDI-1 WW, VLPs were found in electron-dense inclusion bodies in the cytoplasm of transfected cells. In contrast, when cells that coexpressed Gag-Ub and LDI-1 WW were examined, inclusion bodies were detected but did not contain VLPs. These results indicate that the ubiquitination of Gag is dependent upon Nedd4 binding to the L domain and suggest that Nedd4 has additional functions during RSV release besides the ubiquitination of Gag.
Cyclin/CDK Regulates the Nucleocytoplasmic Localization of the Human Papillomavirus E1 DNA Helicase
Authors: Deng Wentao , Lin Biing Yuan , Jin Ge , Wheeler Crystal G. , Ma Tianlin , Harper J. Wade , Broker Thomas R. , Chow Louise T. 
Published: November 2004

ABSTRACT
Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.
Localization and Characterization of Flavivirus Envelope Glycoprotein Cross-Reactive Epitopes
Authors: Crill Wayne D. , Chang Gwong-Jen J. 
Published: November 2004

ABSTRACT
The flavivirus E glycoprotein, the primary antigen that induces protective immunity, is essential for membrane fusion and mediates binding to cellular receptors. Human flavivirus infections stimulate virus species-specific as well as flavivirus cross-reactive immune responses. Flavivirus cross-reactive antibodies in human sera create a serious problem for serodiagnosis, especially for secondary flavivirus infections, due to the difficulty of differentiating primary from secondary cross-reactive serum antibodies. The presence of subneutralizing levels of flavivirus cross-reactive serum antibodies may result in a dramatic increase in the severity of secondary flavivirus infections via antibody-dependent enhancement. An understanding of flavivirus E-glycoprotein cross-reactive epitopes is therefore critical for improving public health responses to these serious diseases. We identified six E-glycoprotein residues that are incorporated into three distinct flavivirus cross-reactive epitopes. Two of these epitopes which are recognized by distinct monoclonal antibodies contain overlapping continuous residues located within the highly conserved fusion peptide. The third epitope consists of discontinuous residues that are structurally related to the strictly conserved tryptophan at dengue virus serotype 2 E-glycoprotein position 231.
Human Metapneumovirus Persists in BALB/c Mice despite the Presence of Neutralizing Antibodies
Authors: Alvarez Rene , Harrod Kevin S. , Shieh Wun-Ju , Zaki Sherif , Tripp Ralph A. 
Published: November 2004

ABSTRACTHuman metapneumovirus (HMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. Recent epidemiological evidence indicates that HMPV may cocirculate with respiratory syncytial virus, and HMPV infection has been associated with other respiratory diseases. In this study, we show that BALB/c mice are susceptible to HMPV infection, the virus replicates in the lungs with biphasic growth kinetics in which peak titers occur at days 7 and 14 postinfection (p.i.), and infectious HMPV can be recovered from lungs up to day 60 p.i. In addition, we show that genomic HMPV RNA can be detected in the lungs for ≥180 days p.i. by reverse transcription-PCR; however, neither HMPV RNA nor infectious virus can be detected in serum, spleen, kidneys, heart, trachea, and brain tissue. Lung histopathology revealed prevalent mononuclear cell infiltration in the interstitium beginning at day 2 p.i. and peaking at day 4 p.i. which decreased by day 14 p.i. and was associated with airway remodeling. Increased mucus production evident at day 2 p.i. was concordant with increased bronchial and bronchiolar inflammation. HMPV-specific antibodies were detected by day 14 p.i., neutralizing antibody titers reached ≥6.46 log2end-point titers by day 28 p.i., and depletion of T cells or NK cells resulted in increased HMPV titers in the lungs, suggesting some immune control of viral persistence. This study shows that BALB/c mice are amenable for HMPV studies and indicates that HMPV persists as infectious virus in the lungs of normal mice for several weeks postinfection.
Definition of Sequence Requirements for Latency-Associated Nuclear Antigen 1 Binding to Kaposi's Sarcoma-Associated Herpesvirus DNA
Authors: Srinivasan Viswanathan , Komatsu Takashi , Ballestas Mary E. , Kaye Kenneth M. 
Published: November 2004

ABSTRACT

In latent infection, Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen 1 (LANA1)-specific binding to KSHV terminal repeat DNA mediates multicopy episome persistence. We now use electrophoretic mobility shift assays to investigate LANA1 binding to its 20-bp cognate sequence. Mutations at positions 6, 7, and 8 (
6
CCC
8
) severely reduced LANA1 binding, whereas mutations at other positions only modestly reduced binding. Since
6
CCC
8
is in the 5′ half of an inverted repeat sequence, these results are consistent with an asymmetric role for the inverted repeat in LANA1 binding.
Overexpression of 7a, a Protein Specifically Encoded by the Severe Acute Respiratory Syndrome Coronavirus, Induces Apoptosis via a Caspase-Dependent Pathway
Authors: Tan Yee-Joo , Fielding Burtram C. , Goh Phuay-Yee , Shen Shuo , Tan Timothy H. P. , Lim Seng Gee , Hong Wanjin 
Published: November 2004

ABSTRACT
Besides genes that are homologous to proteins found in other coronaviruses, the severe acute respiratory syndrome coronavirus genome also contains nine other potential open reading frames. Previously, we have characterized the expression and cellular localization of two of these “accessory” viral proteins, 3a (previously termed U274) and 7a (previously termed U122). In this study, we further examined whether they can induce apoptosis, which has been observed clinically. We showed that the overexpression of 7a, but not of 3a or the viral structural proteins, nucleocapsid, membrane, and envelope, induces apoptosis. 7a induces apoptosis via a caspase-dependent pathway and in cell lines derived from different organs, including lung, kidney, and liver.
A Period of Transient Viremia and Occult Infection Precedes Persistent Viremia and Antiviral Immune Responses during Multiple Low-Dose Intravaginal Simian Immunodeficiency Virus Inoculations
Authors: Ma Zhong-Min , Abel Kristina , Rourke Tracy , Wang Yichuan , Miller Christopher J. 
Published: November 2004

ABSTRACT

In rhesus macaques, classic systemic infection, characterized by persistent viremia and seroconversion, occurred after multiple low-dose (10
3
50% tissue culture infective doses) intravaginal (IVAG) inoculations with simian immunodeficiency virus (SIV) strain SIVmac251. Monkeys developed classic SIV infections after a variable number of low-dose IVAG exposures to SIVmac251. Once established, the systemic infection was identical to SIV infection following high-dose IVAG SIV inoculation. However, occult systemic infection characterized by transient cell-associated or cell-free viremia consistently occurred early in the series of multiple vaginal SIV exposures. Further, antiviral cellular immune responses were present prior to the establishment of a classic systemic infection in the low-dose vaginal SIV transmission model.
Reductions in pre-season training loads reduce training injury rates in rugby league players
Authors: Gabbett T J 
Published: November 2004

Objectives:To investigate if reductions in pre-season training loads reduced the incidence of training injuries in rugby league players, and to determine if the reductions in training loads compromised the improvements in physical fitness obtained during the pre-season preparation period.Methods:A total of 220 sub-elite rugby league players participated in this 3 year prospective study. Players underwent measurements of speed, muscular power, and maximal aerobic power before and after three 4 month (December to March) pre-season preparation periods (2001–2003). A periodised skills and conditioning program was implemented, with training loads progressively increased in the general preparatory phase of the season (December to February) and reduced slightly in March in preparation for the competitive phase of the season. Training loads were calculated by multiplying the training session intensity by the duration of the training session. Following the initial season (2001), training loads were reduced through reductions in training duration (2002) and training intensity (2003). The incidence of injury was prospectively recorded over the three pre-season periods.Results:The training loads for the 2002 and 2003 pre-season periods were significantly lower (p<0.001) than those in 2001. The incidence of injury was significantly higher in the 2001 pre-season than the 2002 and 2003 pre-season periods. The increases in maximal aerobic power progressively improved across the three seasons with a 62–88% probability that the 2002 and 2003 pre-season improvements in maximal aerobic power were of greater physiological significance than the 2001 pre-season improvements in maximal aerobic power.Conclusions:These findings demonstrate that reductions in pre-season training loads reduce training injury rates in rugby league players and result in greater improvements in maximal aerobic power.
Fatal pulmonary embolism in hospitalised patients: a necropsy review
Authors: Alikhan R , Peters F , Wilmott R , Cohen A T 
Published: November 2004

Aims:To carry out a retrospective review of all postmortem reports during the period 1991 to 2000 at King’s College Hospital, London, as an extension of a previous analysis performed for the period 1965 to 1990.Methods:The number of deaths resulting from necropsy confirmed fatal pulmonary embolism in hospitalised patients was determined, and a limited analysis of the clinical characteristics of those patients who died was performed.Results:During the 10 year period, 16 104 deaths occurred and 6833 (42.4%) necropsies were performed. The outcome measure, fatal pulmonary embolism, was recorded as cause of death in 265 cases (3.9% of all necropsies; 5.2% of adult cases). No deaths from pulmonary embolism occurred in patients under 18 years of age; 80.0% occurred in patients older than 60 years. Of the fatal emboli, 214 of 265 (80.8%) occurred in patients who had not undergone recent surgery. Of these patients, 110 (51.4%) had suffered an acute medical illness in the six weeks before death, most often an acute infectious episode (26 cases).Conclusions:Thromboembolic events remain a relatively common cause of death in hospitalised patients and appear to occur more frequently in non-surgical than in surgical patients.