bluuebunny/crossref_metadata_2025_split huggingface dataset:
Altered Proteolysis and Global Gene Expression in Hepatitis B Virus X Transgenic Mouse Liver
Authors: Hu Zongyi , Zhang Zhensheng , Kim Jin Woo , Huang Ying , Liang T. Jake 
Published: January 2006

ABSTRACT
Hepatitis B virus X (HBX) is essential for the productive infection of hepatitis B virus (HBV) in vivo and has a pleiotropic effect on host cells. We have previously demonstrated that the proteasome complex is a cellular target of HBX, that HBX alters the proteolytic activity of proteasome in vitro, and that inhibition of proteasome leads to enhanced viral replication, suggesting that HBX and proteasome interaction plays a crucial role in the life cycle and pathogenesis of HBV. In the present study, we tested the effect of HBX on the proteasome activities in vivo in a transgenic mouse model in which HBX expression is developmentally regulated by the mouse major urinary promoter in the liver. In addition, microarray analysis was performed to examine the effect of HBX expression on the global gene expression profile of the liver. The results showed that the peptidase activities of the proteasome were reduced in the HBX transgenic mouse liver, whereas the activity of another cellular protease was elevated, suggesting a compensatory mechanism in protein degradation. In the microarray analysis, diverse genes were altered in the HBX mouse livers and the number of genes with significant changes increased progressively with age. Functional clustering showed that a number of genes involved in transcription and cell growth were significantly affected in the HBX mice, possibly accounting for the observed pleiotropic effect of HBX. In particular, insulin-like growth factor-binding protein 1 was down-regulated in the HBX mouse liver. The down-regulation was similarly observed during acute woodchuck hepatitis virus infection. Other changes including up-regulation of proteolysis-related genes may also contribute to the profound alterations of liver functions in HBV infection.
Characterization of Antibody Responses Elicited by Human Immunodeficiency Virus Type 1 Primary Isolate Trimeric and Monomeric Envelope Glycoproteins in Selected Adjuvants
Authors: Li Y. , Svehla K. , Mathy N. L. , Voss G. , Mascola J. R. , Wyatt R. 
Published: January 2006

ABSTRACTWe previously reported that soluble, stable YU2 gp140 trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein immunogens could elicit improved breadth of neutralization against HIV-1 isolates compared to monomeric YU2 gp120 proteins. In this guinea pig immunization study, we sought to extend these data and determine if adjuvant could quantitatively or qualitatively alter the neutralizing response elicited by trimeric or monomeric immunogens. Consistent with our earlier studies, the YU2 gp140 immunogens elicited higher-titer neutralizing antibodies against homologous and heterologous isolates than those elicited by monomeric YU2 gp120. Additionally, the GlaxoSmithKline family of adjuvants AS01B, AS02A, and AS03 induced higher levels of neutralizing antibodies compared to emulsification of the same immunogens in Ribi adjuvant. Further analysis of vaccine sera indicated that homologous virus neutralization was not mediated by antibodies to the V3 loop, although V3 loop-directed neutralization could be detected for some heterologous isolates. In most gp120-inoculated animals, the homologous YU2 neutralization activity was inhibited by a peptide derived from the YU2 V1 loop, whereas the neutralizing activity elicited by YU2 gp140 trimers was much less sensitive to V1 peptide inhibition. Consistent with a less V1-focused antibody response, sera from the gp140-immunized animals more efficiently neutralized heterologous HIV-1 isolates, as determined by two distinct neutralization formats. Thus, there appear to be qualitative differences in the neutralizing antibody response elicited by YU2 gp140 compared to YU2 monomeric gp120. Further mapping analysis of more conserved regions of gp120/gp41 may be required to determine the neutralizing specificity elicited by the trimeric immunogens.
A Tick-Borne Langat Virus Mutant That Is Temperature Sensitive and Host Range Restricted in Neuroblastoma Cells and Lacks Neuroinvasiveness for Immunodeficient Mice
Authors: Rumyantsev Alexander A. , Murphy Brian R. , Pletnev Alexander G. 
Published: January 2006

ABSTRACT

Langat virus (LGT), the naturally attenuated member of the tick-borne encephalitis virus (TBEV) complex, was tested extensively in clinical trials as a live TBEV vaccine and was found to induce a protective, durable immune response; however, it retained a low residual neuroinvasiveness in mice and humans. In order to ablate or reduce this property, LGT mutants that produced a small plaque size or temperature-sensitive (
ts
) phenotype in Vero cells were generated using 5-fluorouracil. One of these
ts
mutants, clone E5-104, exhibited a more than 10
3
-fold reduction in replication at the permissive temperature in both mouse and human neuroblastoma cells and lacked detectable neuroinvasiveness for highly sensitive immunodeficient mice. The E5-104 mutant possessed five amino acid substitutions in the structural protein E and one change in each of the nonstructural proteins NS3 and NS5. Using reverse genetics, we demonstrated that a Lys
46
→Glu substitution in NS3 as well as a single Lys
315
→Glu change in E significantly impaired the growth of LGT in neuroblastoma cells and reduced its peripheral neurovirulence for SCID mice. This study and our previous experience with chimeric flaviviruses indicated that a decrease in viral replication in neuroblastoma cells might serve as a predictor of in vivo attenuation of the neurotropic flaviviruses. The combination of seven mutations identified in the nonneuroinvasive E5-104 mutant provided a useful foundation for further development of a live attenuated TBEV vaccine. An evaluation of the complete sequence of virus recovered from brain of SCID mice inoculated with LGT mutants identified sites in the LGT genome that promoted neurovirulence/neuroinvasiveness.
Role of Helix P of the Human Cytomegalovirus DNA Polymerase in Resistance and Hypersusceptibility to the Antiviral Drug Foscarnet
Authors: Tchesnokov Egor P. , Gilbert Christian , Boivin Guy , Götte Matthias 
Published: January 2006

ABSTRACT

Mutations in the human cytomegalovirus DNA polymerase (UL54) can not only decrease but also increase susceptibility to the pyrophosphate (PP
i
) analogue foscarnet. The proximity of L802M, which confers resistance, and K805Q, which confers hypersusceptibility, suggests a possible unifying mechanism that affects drug susceptibility in one direction or the other. We found that the polymerase activities of L802M- and K805Q-containing mutant enzymes were literally indistinguishable from that of wild-type UL54; however, susceptibility to foscarnet was decreased or increased, respectively. A comparison with the crystal structure model of the related RB69 polymerase suggests that L802 and K805 are located in the conserved α-helix P that is implicated in nucleotide binding. Although L802 and K805 do not appear to make direct contacts with the incoming nucleotide, it is conceivable that changes at these residues could exert their effects through the adjacent, highly conserved amino acids Q807 and/or K811. Our data show that a K811A substitution in UL54 causes reductions in rates of nucleotide incorporation. The activity of the Q807A mutant is only marginally affected, while this enzyme shows relatively high levels of resistance to foscarnet. Based on these data, we suggest that L802M exerts its effects through subtle structural changes in α-helix P that affect the precise positioning of Q807 and, in turn, its presumptive involvement in binding of foscarnet. In contrast, the removal of a positive charge associated with the K805Q change may facilitate access or increase affinity to the adjacent Q807.
Structure and Dynamics of Coxsackievirus B4 2A Proteinase, an Enyzme Involved in the Etiology of Heart Disease
Authors: Baxter Nicola J. , Roetzer Andreas , Liebig Hans-Dieter , Sedelnikova Svetlana E. , Hounslow Andrea M. , Skern Tim , Waltho Jonathan P. 
Published: January 2006

ABSTRACT

The 2A proteinases (2A
pro
) from the picornavirus family are multifunctional cysteine proteinases that perform essential roles during viral replication, involving viral polyprotein self-processing and shutting down host cell protein synthesis through cleavage of the eukaryotic initiation factor 4G (eIF4G) proteins. Coxsackievirus B4 (CVB4) 2A
pro
also cleaves heart muscle dystrophin, leading to cytoskeletal dysfunction and the symptoms of human acquired dilated cardiomyopathy. We have determined the solution structure of CVB4 2A
pro
(extending in an N-terminal direction to include the C-terminal eight residues of CVB4 VP1, which completes the VP1-2A
pro
substrate region). In terms of overall fold, it is similar to the crystal structure of the mature human rhinovirus serotype 2 (HRV2) 2A
pro
, but the relatively low level (40%) of sequence identity leads to a substantially different surface. We show that differences in the cI-to-eI2 loop between HRV2 and CVB4 2A
pro
translate to differences in the mechanism of eIF4GI recognition. Additionally, the nuclear magnetic resonance relaxation properties of CVB4 2A
pro
, particularly of residues G1 to S7, F64 to S67, and P107 to G111, reveal that the substrate region is exchanging in and out of a conformation in which it occupies the active site with association and dissociation rates in the range of 100 to 1,000 s
−1
. This exchange influences the conformation of the active site and points to a mechanism for how self-processing can occur efficiently while product inhibition is avoided.
Infectious Molecular Clones from a Simian Immunodeficiency Virus-Infected Rapid-Progressor (RP) Macaque: Evidence of Differential Selection of RP-Specific Envelope Mutations In Vitro and In Vivo
Authors: Kuwata Takeo , Dehghani Houman , Brown Charles R. , Plishka Ronald , Buckler-White Alicia , Igarashi Tatsuhiko , Mattapallil Joseph , Roederer Mario , Hirsch Vanessa M. 
Published: January 2006

ABSTRACT

A minor fraction of simian immunodeficiency virus (SIV)-infected macaques progress rapidly to AIDS in the absence of SIV-specific immune responses. Common mutations in conserved residues of
env
in three SIVsmE543-3-infected rapid-progressor (RP) macaques suggest the evolution of a common viral variant in RP macaques. The goal of the present study was to analyze the biological properties of these variants in vitro and in vivo through the derivation of infectious molecular clones. Virus isolated from a SIVsmE543-3-infected RP macaque, H445 was used to inoculate six naive rhesus macaques. Although RP-specific mutations dominated in H445 tissues, they represented only 10% of the population of the virus stock, suggesting a selective disadvantage in vitro. Only one of these macaques (H635) progressed rapidly to AIDS. Plasma virus during primary infection of H635 was similar to the inoculum. However, RP-specific mutations were apparently rapidly reselected by 4 to 9 weeks postinfection. Terminal plasma from H635 was used as a source of viral RNA to generate seven full-length, infectious molecular clones. With the exception of one clone, which was similar to SIVsmE543-3, clones with RP-specific mutations replicated with delayed kinetics in rhesus peripheral blood mononuclear cells and human T-cell lines. None of the clones replicated in monocyte-derived or alveolar macrophages, and all used CCR5 as their major coreceptor. RP variants appear to be well adapted to replicate in vivo in RP macaques but are at a disadvantage in tissue culture compared to their parent, SIVsmE543-3. Therefore, tissue culture may not provide a good surrogate for replication of RP variants in macaques. These infectious clones will provide a valuable reagent to study the roles of specific viral variants in rapid progression in vivo.
Herpes Simplex Virus 1-Encoded Protein Kinase UL13 Phosphorylates Viral Us3 Protein Kinase and Regulates Nuclear Localization of Viral Envelopment Factors UL34 and UL31
Authors: Kato Akihisa , Yamamoto Mayuko , Ohno Takashi , Tanaka Michiko , Sata Tetsutaro , Nishiyama Yukihiro , Kawaguchi Yasushi 
Published: January 2006

ABSTRACT
UL13 and Us3 are protein kinases encoded by herpes simplex virus 1. We report here that Us3 is a physiological substrate for UL13 in infected cells, based on the following observations. (i) The electrophoretic mobility, in denaturing gels, of Us3 isoforms from Vero cells infected with wild-type virus was slower than that of isoforms from cells infected with a UL13 deletion mutant virus (ΔUL13). After treatment with phosphatase, the electrophoretic mobility of the Us3 isoforms from cells infected with wild-type virus changed, with one isoform migrating as fast as one of the Us3 isoforms from ΔUL13-infected cells. (ii) A recombinant protein containing a domain of Us3 was phosphorylated by UL13 in vitro. (iii) The phenotype of ΔUL13 resembles that of a recombinant virus lacking the Us3 gene (ΔUs3) with respect to localization of the viral envelopment factors UL34 and UL31, whose localization has been shown to be regulated by Us3. UL34 and UL31 are localized in a smooth pattern throughout the nuclei of cells infected with wild-type virus, whereas their localization in ΔUL13- and ΔUs3-infected cells appeared as nuclear punctate patterns. These results indicate that UL13 phosphorylates Us3 in infected cells and regulates UL34 and UL31 localization, either by phosphorylating Us3 or by a Us3-independent mechanism.
Canine Adenovirus Vectors for Lung-Directed Gene Transfer: Efficacy, Immune Response, and Duration of Transgene Expression Using Helper-Dependent Vectors
Authors: Keriel Anne , René Céline , Galer Chad , Zabner Joseph , Kremer Eric J. 
Published: January 2006

ABSTRACTA major hurdle to the successful clinical use of some viral vectors relates to the innate, adaptive, and memory immune responses that limit the efficiency and duration of transgene expression. Some of these drawbacks may be circumvented by using vectors derived from nonhuman viruses such as canine adenovirus type 2 (CAV-2). Here, we evaluated the potential of CAV-2 vectors for gene transfer to the respiratory tract. We found that CAV-2 transduction was efficient in vivo in the mouse respiratory tract, and ex vivo in well-differentiated human pulmonary epithelia. Notably, the in vivo and ex vivo efficiency was poorly inhibited by sera from mice immunized with a human adenovirus type 5 (HAd5, a ubiquitous human pathogen) vector or by human sera containing HAd5 neutralizing antibodies. Following intranasal instillation in mice, CAV-2 vectors also led to a lower level of inflammatory cytokine secretion and cellular infiltration compared to HAd5 vectors. Moreover, CAV-2 transduction efficiency was increased in vitro in human pulmonary cells and in vivo in the mouse respiratory tract by FK228, a histone deacetylase inhibitor. Finally, by using a helper-dependent CAV-2 vector, we increased the in vivo duration of transgene expression to at least 3 months in immunocompetent mice without immunosuppression. Our data suggest that CAV-2 vectors may be efficient and safe tools for long-term clinical gene transfer to the respiratory tract.
High-Resolution Molecular and Antigen Structure of the VP8* Core of a Sialic Acid-Independent Human Rotavirus Strain
Authors: Monnier Nilah , Higo-Moriguchi Kyoko , Sun Zhen-Yu J. , Prasad B. V. Venkataram , Taniguchi Koki , Dormitzer Philip R. 
Published: January 2006

ABSTRACTThe most intensively studied rotavirus strains initially attach to cells when the “heads” of their protruding spikes bind cell surface sialic acid. Rotavirus strains that cause disease in humans do not bind this ligand. The structure of the sialic acid binding head (the VP8* core) from the simian rotavirus strain RRV has been reported, and neutralization epitopes have been mapped onto its surface. We report here a 1.6-Å resolution crystal structure of the equivalent domain from the sialic acid-independent rotavirus strain DS-1, which causes gastroenteritis in humans. Although the RRV and DS-1 VP8* cores differ functionally, they share the same galectin-like fold. Differences between the RRV and DS-1 VP8* cores in the region that corresponds to the RRV sialic acid binding site make it unlikely that DS-1 VP8* binds an alternative carbohydrate ligand in this location. In the crystals, a surface cleft on each DS-1 VP8* core binds N-terminal residues from a neighboring molecule. This cleft may function as a ligand binding site during rotavirus replication. We also report an escape mutant analysis, which allows the mapping of heterotypic neutralizing epitopes recognized by human monoclonal antibodies onto the surface of the VP8* core. The distribution of escape mutations on the DS-1 VP8* core indicates that neutralizing antibodies that recognize VP8* of human rotavirus strains may bind a conformation of the spike that differs from those observed to date.
Two Domains That Control Prefusion Stability and Transport Competence of the Measles Virus Fusion Protein
Authors: Doyle Joshua , Prussia Andrew , White Laura K. , Sun Aiming , Liotta Dennis C. , Snyder James P. , Compans Richard W. , Plemper Richard K. 
Published: January 2006

ABSTRACT
Most viral glycoproteins mediating membrane fusion adopt a metastable native conformation and undergo major conformational changes during fusion. We previously described a panel of compounds that specifically prevent fusion induced by measles virus (MV), most likely by interfering with conformational rearrangements of the MV fusion (F) protein. To further elucidate the basis of inhibition and better understand the mechanism of MV glycoprotein-mediated fusion, we generated and characterized resistant MV variants. Spontaneous mutations conferring drug resistance were confirmed in transient assays and in the context of recombinant virions and were in all cases located in the fusion protein. Several mutations emerged independently at F position 462, which is located in the C-terminal heptad repeat (HR-B) domain. In peptide competition assays, all HR-B mutants at residue 462 revealed reduced affinity for binding to the HR-A core complex compared to unmodified HR-B. Combining mutations at residue 462 with mutations in the distal F head region, which we had previously identified as mediating drug resistance, causes intracellular retention of the mutant proteins. The transport competence and activity of the mutants can be restored, however, by incubation at reduced temperature or in the presence of the inhibitory compounds, indicating that the F escape mutants have a reduced conformational stability and that the inhibitors stabilize a transport-competent conformation of the F trimer. The data support the conclusion that residues located in the head domain of the F trimer and the HR-B region contribute jointly to controlling F conformational stability.
Mutational Analysis of the Herpes Simplex Virus Triplex Protein VP19C
Authors: Adamson Walt E. , McNab David , Preston Valerie G. , Rixon Frazer J. 
Published: January 2006

ABSTRACT
Herpes simplex virus type 1 (HSV-1) capsids have an icosahedral structure with capsomers formed by the major capsid protein, VP5, linked in groups of three by distinctive structures called triplexes. Triplexes are heterotrimers formed by two proteins in a 1:2 stoichiometry. The single-copy protein is called VP19C, and the dimeric protein is VP23. We have carried out insertional and deletional mutagenesis on VP19C and have examined the effects of the mutations on virus growth and capsid assembly. Insertional mutagenesis showed that the N-terminal ∼100 amino acids of the protein, which correspond to a region that is poorly conserved among herpesviruses, are insensitive to disruption and that insertions into the rest of the protein had various effects on virus growth. Some, but not all, severely disabled mutants were compromised in the ability to bind VP23 or VP5. Analysis of deletion mutants revealed the presence of a nuclear localization signal (NLS) near the N terminus of VP19C, and this was mapped to a 33-amino-acid region by fusion of specific sequences to a green fluorescent protein marker. By replacing the endogenous NLS with that from the simian virus 40 large T antigen, we were able to show that the first 45 amino acids of VP19C were not essential for assembly of functional capsids and infectious virus particles. However, removing the first 63 amino acids resulted in formation of aberrant capsids and prevented virus growth, suggesting that the poorly conserved N-terminal sequences have some as-yet-unidentified function.
A Genetic System for Rhesus Monkey Rhadinovirus: Use of Recombinant Virus To Quantitate Antibody-Mediated Neutralization
Authors: Bilello John P. , Morgan Jennifer S. , Damania Blossom , Lang Sabine M. , Desrosiers Ronald C. 
Published: January 2006

ABSTRACT
Rhesus monkey rhadinovirus (RRV), a simian gamma-2 herpesvirus closely related to the Kaposi sarcoma-associated herpesvirus, replicates lytically in cultured rhesus monkey fibroblasts and establishes persistence in B cells. Overlapping cosmid clones were generated that encompass the entire 130-kilobase-pair genome of RRV strain 26-95, including the terminal repeat regions required for its replication. Cloned RRV that was produced by cotransfection of overlapping cosmids spanning the entire RRV26-95 genome replicated with growth kinetics and to titers similar to those of the parental, uncloned, wild-type RRV26-95. Expression cassettes for secreted-engineered alkaline phosphatase (SEAP) and green fluorescent protein (GFP) were inserted upstream of the R1 gene, and the cosmid-based system for RRV genome reconstitution was used to generate replication-competent, recombinant RRV that expressed either the SEAP or GFP reporter gene. Using the SEAP and GFP recombinant RRVs, assays were developed to monitor RRV infection, neutralization, and replication. Heat-inactivated sera from rhesus monkeys that were naturally or experimentally infected with RRV were assayed for their ability to neutralize RRV-SEAP and RRV-GFP infectivity using rhesus monkey fibroblasts. Sera from RRV-positive monkeys, but not RRV-negative monkeys, were consistently able to neutralize RRV infectivity when assayed by the production of SEAP activity or by the ability to express GFP. The neutralizing activity was present in the immunoglobulin fraction. Of the 17 rhesus monkeys tested, sera from rhesus monkey 26-95, i.e., the monkey that yielded the RRV 26-95 isolate, had the highest titer of neutralizing activity against RRV26-95. This cosmid-based genetic system and the reporter virus neutralization assay will facilitate study of the contribution of individual RRV glycoproteins to entry into different cell types, particularly fibroblasts and B cells.