bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rhesus and Human Cytomegalovirus Glycoprotein L Are Required for Infection and Cell-to-Cell Spread of Virus but Cannot Complement Each Other
Authors: Bowman J. Jason , Lacayo Juan C. , Burbelo Peter , Fischer Elizabeth R. , Cohen Jeffrey I. 
Published: December 2010

ABSTRACT
Rhesus cytomegalovirus (RhCMV), the homolog of human cytomegalovirus (HCMV), serves as a model for understanding the pathogenesis of HCMV and for developing candidate vaccines. In order to develop a replication-defective virus as a vaccine candidate, we constructed RhCMV with glycoprotein L (gL) deleted. RhCMV gL was essential for viral replication, and virus with gL deleted could only replicate in cells expressing RhCMV gL. Noncomplementing cells infected with RhCMV with gL deleted released intact, noninfectious RhCMV particles that were indistinguishable from wild-type RhCMV by electron microscopy and could be rescued by treatment of cells with polyethylene glycol. In addition, noncomplementing cells infected with RhCMV with gL deleted produced levels of gB, the major target of neutralizing antibodies, at levels similar to those observed in cells infected with wild-type RhCMV. Since RhCMV and HCMV gL share 53% amino acid identity, we determined whether the two proteins could complement the heterologous virus. Cells transfected with an HCMV bacterial artificial chromosome with gL deleted yielded virus that could replicate in human cells expressing HCMV gL. This is the second HCMV mutant with an essential glycoprotein deleted that has been complemented in cell culture. Finally, we found that HCMV gL could not complement the replication of RhCMV with gL deleted and that RhCMV gL could not complement the replication of HCMV with gL deleted. These data indicate that RhCMV and HCMV gL are both essential for replication of their corresponding viruses and, although the two gLs are highly homologous, they are unable to complement each another.
West Nile Virus Differentially Modulates the Unfolded Protein Response To Facilitate Replication and Immune Evasion
Authors: Ambrose Rebecca L. , Mackenzie Jason M. 
Published: December 2010

ABSTRACT

For intracellular survival it is imperative that viruses have the capacity to manipulate various cellular responses, including metabolic and biosynthetic pathways. The unfolded protein response (UPR) is induced by various external and internal stimuli, including the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Our previous studies have indicated that the replication and assembly of the flavivirus West Nile virus strain Kunjin virus (WNV
KUN
) is intimately associated with the ER. Thus, we sought to determine whether the UPR was induced during WNV
KUN
infection. WNV
KUN
induces UPR signaling during replication, which is coordinated with peak replication. Interestingly, signaling is biased toward the ATF6/IRE-1 arm of the response, with high levels of Xbp-1 activation but negligible eukaryotic translation initiation factor 2α phosphorylation and downstream transcription. We show that the PERK-mediated response may partially regulate replication, since external UPR stimulation had a limiting effect on early replication events and cells deficient for PERK demonstrated increased replication and virus release. Significantly, we show that the WNV
KUN
hydrophobic nonstructural proteins NS4A and NS4B are potent inducers of the UPR, which displayed a high correlation in inhibiting Jak-STAT signaling in response to alpha interferon (IFN-α). Sequential removal of the transmembrane domains of NS4A showed that reducing hydrophobicity decreased UPR signaling and restored IFN-α-mediated activation. Overall, these results suggest that WNV
KUN
can stimulate the UPR to facilitate replication and that the induction of a general ER stress response, regulated by hydrophobic WNV
KUN
proteins, can potentiate the inhibition of the antiviral signaling pathway.
Antiviral RNA Interference Responses Induced by Semliki Forest Virus Infection of Mosquito Cells: Characterization, Origin, and Frequency-Dependent Functions of Virus-Derived Small Interfering RNAs
Authors: Siu Ricky W. C. , Fragkoudis Rennos , Simmonds Peter , Donald Claire L. , Chase-Topping Margo E. , Barry Gerald , Attarzadeh-Yazdi Ghassem , Rodriguez-Andres Julio , Nash Anthony A. , Merits Andres , Fazakerley John K. , Kohl Alain 
Published: December 2010

ABSTRACT

RNA interference (RNAi) is an important mosquito defense mechanism against arbovirus infection. In this paper we study the processes underlying antiviral RNAi in
Aedes albopictus
-derived U4.4 mosquito cells infected with Semliki Forest virus (SFV) (
Togaviridae
;
Alphavirus
). The production of virus-derived small interfering RNAs (viRNAs) from viral double-stranded RNA (dsRNA) is a key event in this host response. dsRNA could be formed by RNA replication intermediates, by secondary structures in RNA genomes or antigenomes, or by both. Which of these dsRNAs is the substrate for the generation of viRNAs is a fundamental question. Here we used deep sequencing of viRNAs and bioinformatic analysis of RNA secondary structures to gain insights into the characteristics and origins of viRNAs. An asymmetric distribution of SFV-derived viRNAs with notable areas of high-level viRNA production (hot spots) and no or a low frequency of viRNA production (cold spots) along the length of the viral genome with a slight bias toward the production of genome-derived viRNAs over antigenome-derived viRNAs was observed. Bioinformatic analysis suggests that hot spots of viRNA production are rarely but not generally associated with putative secondary structures in the SFV genome, suggesting that most viRNAs are derived from replicative dsRNA. A pattern of viRNAs almost identical to those of
A. albopictus
cells was observed for
Aedes aegypti
-derived Aag2 cells, suggesting common mechanisms that lead to viRNA production. Hot-spot viRNAs were found to be significantly less efficient at mediating antiviral RNAi than cold-spot viRNAs, pointing toward a nucleic acid-based viral decoy mechanism to evade the RNAi response.
Differential Effects of Human Immunodeficiency Virus Type 1 Vpu on the Stability of BST-2/Tetherin
Authors: Andrew Amy J. , Miyagi Eri , Strebel Klaus 
Published: December 2010

ABSTRACT
BST-2/CD317/tetherin is a host factor that inhibits the release of HIV-1 and other unrelated viruses. A current model proposes that BST-2 physically tethers virions to the surface of virus-producing cells. The HIV-1-encoded Vpu protein effectively antagonizes the activity of BST-2. How Vpu accomplishes this task remains unclear; however, it is known that Vpu has the ability to down-modulate BST-2 from the cell surface. Here we analyzed the effects of Vpu on BST-2 by performing a series of kinetic studies with HeLa, 293T, and CEMx174 cells. Our results indicate that the surface downregulation of BST-2 is not due to an accelerated internalization or reduced recycling of internalized BST-2 but instead is caused by interference with the resupply of newly synthesized BST-2 from within the cell. While our data confirm previous reports that the high-level expression of Vpu can cause the endoplasmic reticulum (ER)-associated degradation of BST-2, we found no evidence that Vpu targets endogenous BST-2 in the ER in the course of a viral infection. Instead, we found that Vpu acts in a post-ER compartment and increases the turnover of newly synthesized mature BST-2. Our observation that Vpu does not affect the recycling of BST-2 suggests that Vpu does not act directly at the cell surface but may interfere with the trafficking of newly synthesized BST-2 to the cell surface, resulting in the accelerated targeting of BST-2 to the lysosomal compartment for degradation.
Rice Dwarf Viruses with Dysfunctional Genomes Generated in Plants Are Filtered Out in Vector Insects: Implications for the Origin of the Virus
Authors: Pu Yingying , Kikuchi Akira , Moriyasu Yusuke , Tomaru Masatoshi , Jin Yan , Suga Haruhisa , Hagiwara Kyoji , Akita Fusamichi , Shimizu Takumi , Netsu Osamu , Suzuki Nobuhiro , Uehara-Ichiki Tamaki , Sasaya Takahide , Wei Taiyun , Li Yi , Omura Toshihiro 
Published: December 2010

ABSTRACT

Rice dwarf virus
(RDV), with 12 double-stranded RNA (dsRNA) genome segments (S1 to S12), replicates in and is transmitted by vector insects. The RDV-plant host-vector insect system allows us to examine the evolution, adaptation, and population genetics of a plant virus. We compared the effects of long-term maintenance of RDV on population structures in its two hosts. The maintenance of RDV in rice plants for several years resulted in gradual accumulation of nonsense mutations in S2 and S10, absence of expression of the encoded proteins, and complete loss of transmissibility. RDV maintained in cultured insect cells for 6 years retained an intact protein-encoding genome. Thus, the structural P2 protein encoded by S2 and the nonstructural Pns10 protein encoded by S10 of RDV are subject to different selective pressures in the two hosts, and mutations accumulating in the host plant are detrimental in vector insects. However, one round of propagation in insect cells or individuals purged the populations of RDV that had accumulated deleterious mutations in host plants, with exclusive survival of fully competent RDV. Our results suggest that during the course of evolution, an ancestral form of RDV, of insect virus origin, might have acquired the ability to replicate in a host plant, given its reproducible mutations in the host plant that abolish vector transmissibility and viability in nature.
Initiation of Epstein-Barr Virus Lytic Replication Requires Transcription and the Formation of a Stable RNA-DNA Hybrid Molecule at OriLyt
Authors: Rennekamp Andrew J. , Lieberman Paul M. 
Published: December 2010

ABSTRACT

The genetic elements of herpesvirus origins of lytic replication have been characterized in detail; however, much remains to be elucidated concerning their functional role in replication initiation. In the case of the Epstein-Barr virus (EBV), we have found that in addition to the two well-defined critical elements required for lytic replication (the upstream and downstream essential elements, UEE and DEE), the origin of lytic replication (OriLyt) also requires the presence of a GC-rich RNA in
cis
. The
BHLF1
transcript is similar to the essential
K5
transcript identified at the Kaposi's sarcoma-associated herpesvirus OriLyt. We have found that truncation of the
BHLF1
transcript or deletion of the TATA box, but not the putative ATG initiation codon, reduce OriLyt function to background levels. By using an antibody specific for RNA-DNA hybrid molecules, we found the
BHLF1
RNA stably annealed to its DNA template during the early steps of lytic reactivation. Furthermore, expression of human RNase H1, which degrades RNA in RNA-DNA hybrids, drastically reduces OriLyt-dependent DNA replication as well as recruitment of the viral single-stranded DNA binding protein BALF2 to OriLyt. These studies suggest that a GC-rich OriLyt transcript is an important component of gammaherpesvirus lytic origins and is required for initial strand separation and loading of core replication proteins.
Crystal Structure of Human Antibody 2909 Reveals Conserved Features of Quaternary Structure-Specific Antibodies That Potently Neutralize HIV-1
Authors: Changela Anita , Wu Xueling , Yang Yongping , Zhang Baoshan , Zhu Jiang , Nardone Glenn A. , O'Dell Sijy , Pancera Marie , Gorny Miroslaw K. , Phogat Sanjay , Robinson James E. , Stamatatos Leonidas , Zolla-Pazner Susan , Mascola John R. , Kwong Peter D. 
Published: December 2010

ABSTRACT

Monoclonal antibody 2909 belongs to a class of potently neutralizing antibodies that recognize quaternary epitopes on HIV-1. Some members of this class, such as 2909, are strain specific, while others, such as antibody PG16, are broadly neutralizing; all, however, recognize a region on the gp120 envelope glycoprotein that includes two loops (V2 and V3) and forms appropriately only in the oligomeric HIV-1 spike (gp120
3
/gp41
3
). Here we present the crystal structure of 2909 and report structure-function analysis with antibody chimeras composed of 2909 and other members of this antibody class. The 2909 structure was dominated by a heavy-chain third-complementarity-determining region (CDR H3) of 21 residues, which comprised 36% of the combining surface and formed a β-hairpin club extending ∼20 Å beyond the rest of the antibody. Sequence analysis and mass spectrometry identified sites of tyrosine sulfation at the middle and top of CDR H3; substitutions with phenylalanine either ablated (middle substitution) or substantially diminished (top substitution) neutralization. Chimeric antibodies composed of heavy and light chains, exchanged between 2909 and other members of the class, indicated a substantial lack of complementation. Comparison of 2909 to PG16 (which is tyrosine sulfated and the only other member of the class for which a structure has previously been reported) showed that both utilize protruding, anionic CDR H3s for recognition. Thus, despite some diversity, members of this class share structural and functional similarities, with conserved features of the CDR H3 subdomain likely reflecting prevalent solutions by the human immune system for recognition of a quaternary site of HIV-1 vulnerability.
Preferential Induction of Protective T Cell Responses to Theiler's Virus in Resistant (C57BL/6 × SJL)F1 Mice
Authors: Jin Young-Hee , Kang Hyun Seok , Mohindru Mani , Kim Byung S. 
Published: December 2010

ABSTRACTInfection of the central nervous system (CNS) with Theiler's murine encephalomyelitis virus (TMEV) induces an immune-mediated demyelinating disease in susceptible mouse strains such as SJL/J (H-2s) but not in strains such as C57BL/6 (H-2b). In addition, it has been shown that (C57BL/6 × SJL/J)F1 mice (F1 mice), which carry both resistant and susceptible MHC haplotypes (H-2b/s), are resistant to both viral persistence and TMEV-induced demyelinating disease. In this study, we further analyzed the immune responses underlying the resistance of F1 mice. Our study shows that the resistance of F1 mice is associated with a higher level of the initial virus-specific H-2b-restricted CD8+T cell responses than of the H-2s-restricted CD8+T cell responses. In contrast, pathogenic Th17 responses to viral epitopes are lower in F1 mice than in susceptible SJL/J mice. Dominant effects of resistant genes expressed in antigen-presenting cells of F1 mice on regulation of viral replication and induction of protective T cell responses appear to play a crucial role in disease resistance. Although the F1 mice are resistant to disease, the level of viral RNA in the CNS was intermediate between those of SJL/J and C57BL/6 mice, indicating the presence of a threshold of viral expression for pathogenesis.
Roles of VP4 and NSP1 in Determining the Distinctive Replication Capacities of Simian Rotavirus RRV and Bovine Rotavirus UK in the Mouse Biliary Tract
Authors: Feng Ningguo , Sen Adrish , Wolf Marie , Vo Phuoc , Hoshino Yasutaka , Greenberg Harry B. 
Published: December 2010

ABSTRACT

Rotavirus replication and virulence are strongly influenced by virus strain and host species. The rotavirus proteins VP3, VP4, VP7, NSP1, and NSP4 have all been implicated in strain and species restriction of replication; however, the mechanisms have not been fully determined. Simian (RRV) and bovine (UK) rotaviruses have distinctive replication capacities in mouse extraintestinal organs such as the biliary tract. Using reassortants between UK and RRV, we previously demonstrated that the differential replication of these viruses in mouse embryonic fibroblasts is determined by the respective NSP1 proteins, which differ substantially in their abilities to degrade interferon (IFN) regulatory factor 3 (IRF3) and suppress the type I IFN response. In this study, we used an
in vivo
model of rotavirus infection of mouse gallbladder with UK × RRV reassortants to study the genetic and mechanistic basis of systemic rotavirus replication. We found that the low-replication phenotype of UK in biliary tissues was conferred by UK
VP4
and that the high-replication phenotype of RRV was conferred by RRV
VP4
and
NSP1
. Viruses with RRV
VP4
entered cultured mouse cholangiocytes more efficiently than did those with UK
VP4
. Reassortants with RRV
VP4
and UK
NSP1
genes induced high levels of expression of IRF3-dependent p54 in biliary tissues, and their replication was increased 3-fold in IFN-α/β and -γ receptor or STAT1 knockout (KO) mice compared to wild-type mice. Our data indicate that systemic rotavirus strain-specific replication in the murine biliary tract is determined by both viral entry mediated by
VP4
and viral antagonism of the host innate immune response mediated by
NSP1
.
Appraising the Roles of CBLL1 and the Ubiquitin/Proteasome System for Flavivirus Entry and Replication
Authors: Fernandez-Garcia Maria-Dolores , Meertens Laurent , Bonazzi Matteo , Cossart Pascale , Arenzana-Seisdedos Fernando , Amara Ali 
Published: December 2010

ABSTRACT

The ubiquitin ligase CBLL1 (also known as HAKAI) has been proposed to be a critical cellular factor exploited by West Nile virus (WNV) for productive infection. CBLL1 has emerged as a major hit in a recent RNA interference screen designed to identify cellular factors required for the early stages of the WNV life cycle. Follow-up experiments showed that HeLa cells knocked down for CBLL1 by a small interfering RNA (siRNA) failed to internalize WNV particles and resisted infection. Furthermore, depletion of a free-ubiquitin pool by the proteasome inhibitor MG132 abolished WNV endocytosis, suggesting that CBLL1 acts in concert with the ubiquitin proteasome system to mediate virus internalization. Here, we examined the effect of CBLL1 knockdown and proteasome inhibitors on infection by WNV and other flaviviruses. We identified new siRNAs that repress the CBLL1 protein and strongly inhibit the endocytosis of
Listeria monocytogenes
, a bacterial pathogen known to require CBLL1 to invade host cells. Strikingly, however, we detected efficient WNV, dengue virus, and yellow fever virus infection of human cells, despite potent downregulation of CBLL1 by RNA interference. In addition, we found that the proteasome inhibitors MG132 and lactacystin did not affect WNV internalization but strongly repressed flavivirus RNA translation and replication. Together, these data do not support a requirement for CBLL1 during flavivirus entry and rather suggest an essential role of the ubiquitin/proteasome pathway for flavivirus genome amplification.
Results of the UK NEW START Sentinel Node Biopsy Training Program: A Model for Future Surgical Training.
Authors: Goyal A. , MacNeill F. , Newcombe R. , Townson J. , Keshtgar M. , Mansel R. ,   NEW START Regional Trainers and Steering Group
Published: December 2010

Abstract
Background: NEW START the world's largest structured, multi-professional and validated surgical training programme was established to allow rapid transfer of appropriate knowledge and technical skills to ensure safe and competent sentinel node biopsy (SNB) in the UK. The number of training cases is larger than the number of cases in the NSABP-32 randomised trial.Methods: A sentinel node (SN) localisation protocol was standardised using the combined technique of isotope (0.05-0.1ml 99mTc-albumin colloid - Nanocoll®) and blue dye (Patent Blue V) injected into the tumour quadrant peri-areolar tissue. Isotope was injected intra-dermally and static scintigraphic images obtained, blue dye was injected sub-dermally after anaesthetic induction.Multi-professional teams attended a theory/skills-lab day at a regional center. All surgeons then performed 30 consecutive SNB's (minimum 10 node positive) either followed immediately by standard axillary staging - mentorship model - or as a stand-alone procedure - apprenticeship model. An accredited NEW START trainer supervised the first 5 cases, or all 30 cases if stand-alone apprenticeship, in the participant's hospital.Validation standards were based on published literature with a localization rate of &gt;90% and false-negative rate of &lt;10%. Participants continued in validation until the standards were achieved.Results: From October 2004 to December 2008, SNB was performed on 6685 patients, in 103 centres by 210 SNB naive surgeons. SNB was followed by immediate axillary staging (mentorship model) in 87% (5849/6685), axillary nodal metastases were found in 31% (2098/6685) of whom 1821 had SNB followed by immediate axillary staging.Scintigraphy identified axillary lymph node drainage in 85% (5564/6511) with a total SN localisation rate of 99% (6610/6685, 95% CI 0.9% to 1.4%). There were 163 false negatives, resulting in a false negative rate (FNR) of 8.9% (163/1821, 95% CI 7.7% to 10.4%). The median SN yield was 2.0 (range 1-11).Localization and FNR's improved with surgeon caseload but no significant learning curve was identified. The FNR decreased to &lt;10% after 20 cases.Failed localisation rate and false-negative rate by surgeon case number Surgeon Case Number 1 to 1011 to 2021 to 3031 to 4041 to 75Failed localisation rate (%)1.21.11.11.20.9False-negative rate (%)9.410.28.57.76.4 Conclusion: NEW START is a unique training programme that establishes a benchmark for future surgical training. It demonstrates that carefully planned multi-professional training can translate specialist performance standards across a national service and abolish learning curves so ensuring patient safety during training.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1001.