bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pretreatment tumour volume measurement on high‐resolution magnetic resonance imaging as a predictor of survival in cervical cancer
Authors: Soutter W. Patrick , Hanoch Joseph , D'Arcy Tom , Dina Roberto , McIndoe G. Angus , DeSouza Nandita M. 
Published: December 2004

Objective  To evaluate pretreatment tumour volume as a predictor of survival in patients with cervical cancer using both endovaginal and external coil magnetic resonance imaging in order to achieve high spatial resolution and delineate small volume disease.Design  A retrosfection case series.Setting  A tertiary referral centre for gynaecological oncology.Population/Sample  One hundred and six consecutive women with invasive carcinoma of the cervix underwent endovaginal and external coil magnetic resonance imaging on a 0.5‐T or 1.5‐T scanner.Methods  T2‐W FSE images, sagittal and transverse to the cervix, were obtained and tumour volume was calculated on the sagittal images by the standard technique of multiplying the sum of the areas by the slice thickness. Patients were treated in accordance with normal clinical practice and their subsequent outcome was recorded. The relationships between clinical or imaging parameters and survival were assessed with Cox's proportional hazard method.Main outcome measures  Disease‐free survival.Results  In 89 of these women, the tumour was Stage I and 88 of the 106 were treated principally by surgery. The median tumour volume was 4.75 cm3 (upper and lower quartiles 22 and 0.6). The median length of follow up of surviving patients was 223 weeks (quartiles 158 and 274 weeks). Stage, treatment type, lymphovascular space involvement, invasion of the parametrium, closeness of the excision margin, lymph node metastases, and magnetic resonance imaging measurements of tumour volume, parametrial invasion and lymph node disease were all significantly associated with survival in univariate analysis. Only magnetic resonance imaging measurement of tumour volume remained consistently and strongly associated with survival after multivariate analysis of parameters available prior to treatment (P= 0.001, Wald statistic 10.74). A receiver operating characteristic curve of tumour volume and disease‐free survival confirmed the utility of this investigation and suggested that a cutoff around 13.0 cm3 would predict survival with a positive predictive value of 0.93 and a negative predictive value of 0.75.Conclusion  Magnetic resonance imaging assessment of tumour volume using both an endovaginal and an external coil approach provides an accurate prediction of prognosis in cervical cancer and defines a population of women at high risk of recurrence and death. The predictive value of this investigation is superior to the clinical and histological parameters previously used. Use of this technique permits a more accurate choice of treatment options. These results suggest that it is the size of tumour burden that determines the outcome rather than invasion beyond the anatomical margins of the uterus.
In Vitro and In Vivo Mutational Analysis of the 3′-Terminal Regions of Hepatitis E Virus Genomes and Replicons
Authors: Graff Judith , Nguyen Hanh , Kasorndorkbua Chaiyan , Halbur Patrick G. , St. Claire Marisa , Purcell Robert H. , Emerson Suzanne U. 
Published: December 2004

ABSTRACT
Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3′-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3′-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3′-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3′ termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3′ terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3′-terminal mutations in vivo. Incorporation of the 3′-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.
Poliovirus Escape from RNA Interference: Short Interfering RNA-Target Recognition and Implications for Therapeutic Approaches
Authors: Gitlin Leonid , Stone Jeffrey K. , Andino Raul 
Published: December 2004

ABSTRACT
Short interfering RNAs (siRNAs) directed against poliovirus and other viruses effectively inhibit viral replication. Although RNA interference (RNAi) may provide the basis for specific antiviral therapies, the limitations of RNAi antiviral strategies are ill defined. Here, we show that poliovirus readily escapes highly effective siRNAs through unique point mutations within the targeted regions. Competitive analysis of the escape mutants provides insights into the basis of siRNA recognition. The RNAi machinery can tolerate mismatches but is exquisitely sensitive to mutations within the central region and the 3′ end of the target sequence. Indeed, specific mutations in the target sequence resulting in G:U mismatches are sufficient for the virus to escape siRNA inhibition. However, using a pool of siRNAs to simultaneously target multiple sites in the viral genome prevents the emergence of resistant viruses. Our study uncovers the elegant precision of target recognition by the RNAi machinery and provides the basis for the development of effective RNAi-based therapies that prevent viral escape.
Deletion of Penton RGD Motifs Affects the Efficiency of both the Internalization and the Endosome Escape of Viral Particles Containing Adenovirus Serotype 5 or 35 Fiber Knobs
Authors: Shayakhmetov Dmitry M. , Eberly Andrea M. , Li Zong-Yi , Lieber André 
Published: December 2004

ABSTRACTAdenovirus (Ad) vectors are widely used for gene delivery in vitro and in vivo. A solid understanding of the biology of this virus is imperative for the development of novel, effective, and safe vectors. For the group C adenovirus serotypes 2 and 5 that use CAR as a primary attachment receptor, it is known that the penton base RGD motifs interact with cellular integrins and that this interaction promotes virus internalization. However, the RGD motif's impact on the efficiency of postinternalization steps, such as the escape of the virus particle from the endosome, is less defined. Furthermore, the role of penton-integrin interactions remains unknown for new vectors possessing group B Ad fiber knobs that use CD46 as a primary virus attachment receptor. In this study, we used vectors with the RGD motif deleted that contained Ad5 and B-group Ad35 fiber knobs and long fiber shafts and studied the role of RGD-integrin interactions in virus internalization and endosome escape. The deletion of the RGD motif in the penton base did not affect virus attachment, regardless of the type of cellular receptor used for attachment. RGD motif deletion, however, significantly reduced the rate of virus internalization for both the Ad5 and Ad35 fiber knob-containing vectors. This study also demonstrates the role of penton RGD motifs in facilitating the endosome escape step of virus infection and indicates that penton-integrin interactions are involved in internalization of capsid-chimeric CD46-interacting Ads with long fiber shafts.
Spread of Vaccine-Derived Poliovirus from a Paralytic Case in an Immunodeficient Child: an Insight into the Natural Evolution of Oral Polio Vaccine
Authors: Cherkasova E. A. , Yakovenko M. L. , Rezapkin G. V. , Korotkova E. A. , Ivanova O. E. , Eremeeva T. P. , Krasnoproshina L. I. , Romanenkova N. I. , Rozaeva N. R. , Sirota L. , Agol V. I. , Chumakov K. M. 
Published: December 2004

ABSTRACT
Sabin strains used in the manufacture of oral polio vaccine (OPV) replicate in the human organism and can give rise to vaccine-derived polioviruses. The increased neurovirulence of vaccine derivatives has been known since the beginning of OPV use, but their ability to establish circulation in communities has been recognized only recently during the latest stages of the polio eradication campaign. This important observation called for studies of their emergence and evolution as well as extensive surveillance to determine the scope of this phenomenon. Here, we present the results of a study of vaccine-derived isolates from an immunocompromised poliomyelitis patient, the contacts, and the local sewage. All isolates were identified as closely related and slightly evolved vaccine derivatives with a recombinant type 2/type 1 genome. The strains also shared several amino acid substitutions including a mutation in the VP1 protein that was previously shown to be associated with the loss of attenuation. Another mutation in the VP3 protein resulted in altered immunological properties of the isolates, possibly facilitating virus spread in immunized populations. The patterns and rates of the accumulation of synonymous mutations in isolates collected from the patient over the extended period of excretion suggest either a substantially nonuniform rate of mutagenesis throughout the genome, or, more likely, the strains may have been intratypic recombinants between coevolving derivatives with different degrees of divergence from the vaccine parent. This study provides insight into the early stages of the establishment of circulation by runaway vaccine strains.
Vaccinia Virus F1L Protein Is a Tail-Anchored Protein That Functions at the Mitochondria To Inhibit Apoptosis
Authors: Stewart Tara L. , Wasilenko Shawn T. , Barry Michele 
Published: December 2004

ABSTRACT

Members of the poxvirus family encode multiple immune evasion proteins, including proteins that regulate apoptosis. We recently identified one such protein, F1L, encoded by vaccinia virus, the prototypic member of the poxvirus family. F1L localizes to the mitochondria and inhibits apoptosis by interfering with the release of cytochrome
c
, the pivotal commitment step in the apoptotic cascade. Sequence analysis of the F1L open reading frame revealed a C-terminal motif composed of a 12-amino-acid transmembrane domain flanked by positively charged lysines, followed by an 8-amino-acid hydrophilic tail. By generating a series of F1L deletion constructs, we show that the C-terminal domain is necessary and sufficient for localization of F1L to the mitochondria. In addition, mutation of lysines 219 and 222 downstream of the C-terminal transmembrane domain resulted in altered localization of F1L to the endoplasmic reticulum. Using F1L protein generated in an in vitro transcription-translation system, we found that F1L was posttranslationally inserted into mitochondria and tightly associated with mitochondrial membranes as demonstrated by resistance to alkaline extraction. Sensitivity to protease digestion showed that the N terminus of F1L was exposed to the cytoplasm. Utilizing various F1L deletion constructs, we found that F1L localization to the mitochondria was necessary to inhibit apoptosis, since constructs that no longer localized to the mitochondria had reduced antiapoptotic ability. Our studies show that F1L is a new member of the tail-anchored protein family that localizes to mitochondria during virus infection and inhibits apoptosis as a means to enhance virus survival.
Inhibition of Human Papillomavirus Type 16 E7 Phosphorylation by the S100 MRP-8/14 Protein Complex
Authors: Tugizov Sharof , Berline Jennifer , Herrera Rossana , Penaranda Maria Elena , Nakagawa Mayumi , Palefsky Joel 
Published: December 2004

ABSTRACT

The human papillomavirus type 16 (HPV16) E7 is a major viral oncoprotein that is phosphorylated by casein kinase II (CKII). Two S100 family calcium-binding proteins, macrophage inhibitory-related factor protein 8 (MRP-8) and MRP-14, form a protein complex, MRP-8/14, that inactivates CKII. The MRP-8/14 protein complex may inhibit CKII-mediated E7 phosphorylation and therefore may alter its interaction with cellular ligands and reduce E7 oncogenic activity. We examined the inhibitory effect of the MRP-8/14 complex on CKII activity and HPV16 E7 phosphorylation. We have shown that CKII activity and HPV16 E7 phosphorylation were inhibited by uptake of exogenous MRP-8/14 and activation of endogenous MRP-8/14. MRP-8/14-mediated inhibition of E7 phosphorylation occurred at the G
1
phase of the cell cycle. Analysis of MRP expression in primary keratinocytes and in HPV16- and 18-transformed cervical and foreskin epithelial cell lines showed that expression of MRP-8, MRP-14, and the MRP-8/14 complex was detected only in primary untransformed keratinocytes and not in the HPV-infected immortalized epithelial cells. CKII activity in HPV-immortalized keratinocytes was approximately fourfold higher than in HPV-negative primary keratinocytes. Treatment of HPV-positive immortalized epithelial cells with exogenous MRP-8/14 resulted in E7 hypophosphorylation and complete inhibition of cell growth within 2 weeks, compared with HPV-negative primary and immortalized HPV-negative cervical epithelial cells, which showed 25 and 40% growth inhibition, respectively. Together these results suggests that the MRP-8/14 protein complex in HPV-infected epithelial cells may play an important role in regulation of CKII-mediated E7 phosphorylation and inhibition of its oncogenic activity.
Roles of Carboxyl-Terminal and Farnesylated Residues in the Functions of the Large Hepatitis Delta Antigen
Authors: O'Malley Brendan , Lazinski David W. 
Published: December 2004

ABSTRACT
The large hepatitis delta antigen (HDAg-L) mediates hepatitis delta virus (HDV) assembly and inhibits HDV RNA replication. Farnesylation of the cysteine residue within the HDAg-L carboxyl terminus is required for both functions. Here, HDAg-L proteins from different HDV genotypes and genotype chimeric proteins were analyzed for their ability to incorporate into virus-like particles (VLPs). Observed differences in efficiency of VLP incorporation could be attributed to genotype-specific differences within the HDAg-L carboxyl terminus. Using a novel assay to quantify the extent of HDAg-L farnesylation, we found that genotype 3 HDAg-L was inefficiently farnesylated when expressed in the absence of the small hepatitis delta antigen (HDAg-S). However, as the intracellular ratio of HDAg-S to HDAg-L was increased, so too was the extent of HDAg-L farnesylation for all three genotypes. Single point mutations within the carboxyl terminus of HDAg-L were screened, and three mutants that severely inhibited assembly without affecting farnesylation were identified. The observed assembly defects persisted under conditions where the mutants were known to have access to the site of VLP assembly. Therefore, the corresponding residues within the wild-type protein are likely required for direct interaction with viral envelope proteins. Finally, it was observed that when HDAg-S was artificially myristoylated, it could efficiently inhibit HDV RNA replication. Hence, a general association with membranes enables HDAg to inhibit replication. In contrast, although myristoylated HDAg-S was incorporated into VLPs far more efficiently than HDAg-S or nonfarnesylated HDAg-L, it was incorporated far less efficiently than wild-type HDAg-L; thus, farnesylation was required for efficient assembly.
Mass Spectroscopic Characterization of the Coronavirus Infectious Bronchitis Virus Nucleoprotein and Elucidation of the Role of Phosphorylation in RNA Binding by Using Surface Plasmon Resonance
Authors: Chen Hongying , Gill Andrew , Dove Brian K. , Emmett Stevan R. , Kemp C. Fred , Ritchie Mark A. , Dee Michael , Hiscox Julian A. 
Published: December 2004

ABSTRACTPhosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphosphorylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5′ end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.
Loss and Gain of Elicitor Function of
Soybean Mosaic Virus
G7 Provoking
Rsv1
-Mediated Lethal Systemic Hypersensitive Response Maps to P3
Authors: Hajimorad M. R. , Eggenberger A. L. , Hill J. H. 
Published: December 2004

ABSTRACT

Rsv1
, a single dominant resistance gene in soybean PI 96983 (
Rsv1
), confers extreme resistance against all known American strains of
Soybean mosaic virus
(SMV), except G7 and G7d. SMV-G7 provokes a lethal systemic hypersensitive response (LSHR), whereas SMV-G7d, an experimentally evolved variant of SMV-G7, induces systemic mosaic. To identify the elicitor of
Rsv1
-mediated LSHR, chimeras were constructed by exchanging fragments between the molecularly cloned SMV-G7 (pSMV-G7) and SMV-G7d (pSMV-G7d), and their elicitor functions were assessed on PI 96983 (
Rsv1
). pSMV-G7-derived chimeras containing only P3 of SMV-G7d lost the elicitor function, while the reciprocal chimera of pSMV-G7d gained the function. The P3 regions of the two viruses differ by six nucleotides, of which two are translationally silent. The four amino acid differences are located at positions 823, 915, 953, and 1112 of the precursor polypeptide. Analyses of the site-directed point mutants of both the viruses revealed that nucleotide substitutions leading to translationally silent mutations as well as reciprocal amino acid substitution at position 915 did not influence the loss or gain of the elicitor function. pSMV-G7-derived mutants with amino acid substitutions at any of the other three positions lost the ability to provoke LSHR but induced SHR instead. Two concomitant amino acid substitutions at positions 823 (V to M) and 953 (K to E) abolished pSMV-G7 elicitor function, provoking
Rsv1
-mediated SHR. Conversely, pSMV-G7d gained the elicitor function of
Rsv1
-mediated LSHR by a single amino acid substitution at position 823 (M to V), and mutants with amino acid substitutions at position 953 or 1112 induced SHR instead of mosaic. Taken together, the data suggest that strain-specific P3 of SMV is the elicitor of
Rsv1
-mediated LSHR.
Phosphorylation Site Mutations Affect Herpes Simplex Virus Type 1 ICP0 Function
Authors: Davido David J. , von Zagorski William F. , Lane William S. , Schaffer Priscilla A. 
Published: December 2004

ABSTRACT

The herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein infected-cell protein 0 (ICP0) is a strong and global transactivator of both viral and cellular genes. In a previous study, we reported that ICP0 is highly phosphorylated and contains at least seven distinct phosphorylation signals as determined by phosphotryptic peptide mapping (D. J. Davido et al., J. Virol.
76:
1077-1088, 2002). Since phosphorylation affects the activities of many viral regulatory proteins, we sought to determine whether the phosphorylation of ICP0 affects its functions. To address this question, it was first necessary to identify the regions of ICP0 that are phosphorylated. For this purpose, ICP0 was partially purified, and phosphorylation sites were mapped by microcapillary high-pressure liquid chromatography tandem mass spectrometry. Three phosphorylated regions containing 11 putative phosphorylation sites, all within or adjacent to domains important for the transactivating activity of ICP0, were identified. The 11 sites were mutated to alanine as clusters in each of the three regions by site-directed mutagenesis, generating plasmids expressing mutant forms of ICP0: Phos 1 (four mutated sites), Phos 2 (three mutated sites), and Phos 3 (four mutated sites). One-dimensional phosphotryptic peptide analysis confirmed that the phosphorylation state of each Phos mutant form of ICP0 is altered relative to that of wild-type ICP0. In functional assays, the ICP0 phosphorylation site mutations affected the subcellular and subnuclear localization of ICP0, its ability to alter the staining pattern of the nuclear domain 10 (ND10)-associated protein PML, and/or its transactivating activity in Vero cells. Only mutations in Phos 1, however, impaired the ability of ICP0 to complement the replication of an ICP0 null mutant in Vero cells. This study thus suggests that phosphorylation is an important regulator of ICP0 function.
Factor-Independent Assembly of Elongation-Competent Ribosomes by an Internal Ribosome Entry Site Located in an RNA Virus That Infects Penaeid Shrimp
Authors: Cevallos Randal C. , Sarnow Peter 
Published: December 2004

ABSTRACT

The Taura syndrome virus (TSV), a member of the
Dicistroviridae
family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNA
met
. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.