bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hantavirus N Protein Exhibits Genus-Specific Recognition of the Viral RNA Panhandle
Authors: Mir M. A. , Brown B. , Hjelle B. , Duran W. A. , Panganiban A. T. 
Published: October 2006

ABSTRACT

A key genomic characteristic that helps define
Hantavirus
as a genus of the family
Bunyaviridae
is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into “panhandle” hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5′ and 3′ ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera
Bunyavirus
,
Tospovirus
, and
Phlebovirus
or
Nairovirus
. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus
Hantavirus
but not while interacting with panhandles from viruses of other genera of the family
Bunyaviridae
. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus
Hantavirus
.
Multivalent Sequence Recognition by Epstein-Barr Virus Zta Requires Cysteine 171 and an Extension of the Canonical B-ZIP Domain
Authors: Wang Pu , Day Latasha , Lieberman Paul M. 
Published: October 2006

ABSTRACT
Epstein-Barr virus (EBV) immediate-early protein Zta is a member of the basic-leucine zipper (B-ZIP) family of DNA binding proteins that has an unusual capacity to recognize multiple DNA recognition sites, including AP-1 and C/EBP binding sites. To better understand the structure and function of Zta, we have mutagenized cysteine residues within or adjacent to the B-ZIP domain. We found that serine substitution for cysteine 171 (C171S), which lies outside and amino terminal to the B-ZIP basic region, completely abrogates Zta capacity to initiate lytic cycle replication. C171S disrupted Zta transcription activation function of several EBV lytic cycle promoters, including the BMRF1 gene (EA-D) and the other lytic activator, Rta. Overexpression of Rta could not rescue the C171S defect for transcription reactivation or viral DNA replication. Zta C171S was defective for binding to these promoters in vivo, as measured by chromatin immunoprecipitation assay. Purified Zta C171S bound AP-1 sites similar to wild-type Zta, but it was incapable of binding several degenerate Zta sites, including a consensus C/EBP site. Zta truncation mutations reveal that residues N terminal to the B-ZIP (amino acids 156 to 178) confer C/EBP binding capacity to the otherwise AP-1-restricted DNA recognition function. Comparison among viral orthologues of Zta suggest that a conserved N-terminal extension of the consensus B-ZIP domain is required for this multivalent DNA recognition capacity of Zta and is essential for viral reactivation.
Global Structural Changes in Hepatitis B Virus Capsids Induced by the Assembly Effector HAP1
Authors: Bourne Christina R. , Finn M. G. , Zlotnick Adam 
Published: October 2006

ABSTRACT
Hepatitis B virus (HBV) is a leading cause of liver disease and hepatocellular carcinoma; over 400 million people are chronically infected with HBV. Specific anti-HBV treatments, like most antivirals, target enzymes that are similar to host proteins. Virus capsid protein has no human homolog, making its assembly a promising but undeveloped therapeutic target. HAP1 [methyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxylate], a heteroaryldihydropyrimidine, is a potent HBV capsid assembly activator and misdirector. Knowledge of the structural basis for this activity would directly benefit the development of capsid-targeting therapeutic strategies. This report details the crystal structures of icosahedral HBV capsids with and without HAP1. We show that HAP1 leads to global structural changes by movements of subunits as connected rigid bodies. The observed movements cause the fivefold vertices to protrude from the liganded capsid, the threefold vertices to open, and the quasi-sixfold vertices to flatten, explaining the effects of HAP1 on assembled capsids and on the assembly process. We have identified a likely HAP1-binding site that bridges elements of secondary structure within a capsid-bound monomer, offering explanation for assembly activation. This site also interferes with interactions between capsid proteins, leading to quaternary changes and presumably assembly misdirection. These results demonstrate the plasticity of HBV capsids and the molecular basis for a tenable antiviral strategy.
Epstein-Barr Virus Represses the FoxO1 Transcription Factor through Latent Membrane Protein 1 and Latent Membrane Protein 2A
Authors: Shore Angharad M. , White Paul C. , Hui Rosaline C.-Y. , Essafi Abdelkader , Lam Eric W.-F. , Rowe Martin , Brennan Paul 
Published: October 2006

ABSTRACT

Epstein-Barr virus (EBV) infection is associated with the development of many B-cell lymphomas, including Burkitt's lymphoma, Hodgkin's lymphoma, and posttransplant lymphoproliferative disease. The virus alters a diverse range of cellular molecules, which leads to B-cell growth and immortalization. This study was initiated to investigate the interplay between EBV and a proapoptotic transcription factor target, FoxO1. In this report, we show that EBV infection of B cells leads to the downregulation of FoxO1 expression by phosphatidylinositol 3-kinase-mediated nuclear export, by inhibition of FoxO1 mRNA expression, and by alteration of posttranslational modifications. This repression directly correlates with the expression of the FoxO1 target gene
Bcl-6
and inversely correlates with the FoxO1-regulated gene
Cyclin D2
. Expression of the EBV genes for latent membrane protein 1 and latent membrane protein 2A decreases FoxO1 expression. Thus, our data elucidate distinct mechanisms for the regulation of the proapoptotic transcription factor FoxO1 by EBV.
Phospholipase A2 Activity-Dependent Stimulation of Ca
2+
Entry by Human Parvovirus B19 Capsid Protein VP1
Authors: Lupescu Adrian , Bock C.-Thomas , Lang Philipp A. , Aberle Susanne , Kaiser Heike , Kandolf Reinhard , Lang Florian 
Published: October 2006

ABSTRACT

Recent reports demonstrated an association of human parvovirus B19 with inflammatory cardiomyopathy (iCMP), which is accompanied by endothelial dysfunction. As intracellular Ca
2+
activity is a key regulator of cell function and participates in mechanisms leading to endothelial dysfunction, the present experiments explored the effects of the B19 capsid proteins VP1 and VP2. A secreted phospholipase A2 (PLA2)-like activity has been located in the VP1 unique region of the B19 minor capsid protein. As PLA2 has recently been shown to activate the store-operated or capacitative Ca
2+
channel I
CRAC
, we analyzed the impact of the viral PLA2 motif on Ca
2+
entry. We cloned the VP1 and VP2 genes isolated from a patient suffering from fatal B19 iCMP into eukaryotic expression vectors. We also generated a B19 replication-competent plasmid to demonstrate PLA2 activity under the control of the complete B19 genome. After the transfection of human endothelial cells (HMEC-1), cytosolic Ca
2+
activity was determined by utilizing Fura-2 fluorescence. VP1 and VP2 expression did not significantly modify basal cytosolic Ca
2+
activity or the decline of cytosolic Ca
2+
activity following the removal of extracellular Ca
2+
. However, expression of VP1 and of the full-length B19 clone, but not of VP2, significantly accelerated the increase of cytosolic Ca
2+
activity following the readdition of extracellular Ca
2+
in the presence of thapsigargin, indicating an activation of I
CRAC.
The effect of VP1 was mimicked by the PLA2 product lysophosphatidylcholine and abolished by an inactivating mutation of the PLA2-encoding region of the VP1 gene. Our observations point to the activation of Ca
2+
entry by VP1 PLA2 activity, an effect likely participating in the pathophysiology of B19 infection.
Adeno-Associated Virus Type 2 Capsids with Externalized VP1/VP2 Trafficking Domains Are Generated prior to Passage through the Cytoplasm and Are Maintained until Uncoating Occurs in the Nucleus
Authors: Sonntag Florian , Bleker Svenja , Leuchs Barbara , Fischer Roger , Kleinschmidt Jürgen A. 
Published: October 2006

ABSTRACTCommon features of parvovirus capsids are open pores at the fivefold symmetry axes that traverse the virion shell. Upon limited heat treatment in vitro, the pores can function as portals to externalize VP1/VP2 protein N-terminal sequences which harbor infection-relevant functional domains, such as a phospholipase A2catalytic domain. Here we show that adeno-associated virus type 2 (AAV2) also exposes its VP1/VP2 N termini in vivo during infection, presumably in the endosomal compartment. This conformational change is influenced by treatment with lysosomotropic reagents. While incubation of cells with bafilomycin A1 reduced exposure of VP1/VP2 N termini, incubation with chloroquine stimulated externalization transiently. N-terminally located basic amino acid clusters with nuclear localization activity also become exposed in this process and are accessible on the virus capsid when it enters the cytoplasm. This is an obligatory step in AAV2 infection. However, a direct role of these sequences in nuclear translocation of viral capsids could not be determined by microinjection of wild-type or mutant viruses. This suggests that further modifications of the capsid have to take place in a precytoplasmic entry step that prepares the virus for nuclear entry. Microinjection of several capsid-specific antibodies into the cell nucleus blocked AAV2 infection completely, supporting the conclusion that AAV2 capsids bring the infectious genome into the nucleus.
Characterization of the Functional Activities of the Bovine Papillomavirus Type 1 E2 Protein Single-Chain Heterodimers
Authors: Kurg Reet , Tekkel Helena , Abroi Aare , Ustav Mart 
Published: October 2006

ABSTRACT
Papillomaviruses are small DNA viruses which establish persistent infection in the epithelial tissue of various animal species. Three papillomavirus proteins encoded by the bovine papillomavirus type 1 E2 open reading frame have a common C-terminal DNA binding and dimerization domain and function as dimeric proteins in the regulation of viral gene expression, genome replication, and maintenance. The full-length E2 protein, expressed usually at the lowest level of the three, is an activator, while shorter forms of E2, lacking the transactivation domain, serve as repressors of replication and transcription. In virally infected cells, the full-length E2 protein forms heterodimers with repressor forms of the E2 protein and the biological activities of such heterodimers are poorly known. In order to study the functionality of E2 heterodimers, we joined the full-length E2 protein and E2 repressor by a flexible polypeptide hinge so that they formed a single-chain intramolecular dimer. The single-chain E2 heterodimers folded correctly to form genuine pseudodimers capable of binding to the specific E2 protein binding site with high affinity. Characterization of the activities of this protein in transcription showed that it functions as an effective transcriptional activator, which is comparable to what was found for the full-length E2 protein. The single-chain heterodimer is dependent to some extent on Brd4 protein and is able to support papillomavirus origin replication; however, it does not support the partitioning of the multimeric E2 binding site containing plasmids in dividing cells. Our results suggest that E2 heterodimers serve as activators of transcription and replication during the viral life cycle.
Tracking of Peptide-Specific CD4+T-Cell Responses after an Acute Resolving Viral Infection: a Study of Parvovirus B19
Authors: Kasprowicz Victoria , Isa Adiba , Tolfvenstam Thomas , Jeffery Katie , Bowness Paul , Klenerman Paul 
Published: October 2006

ABSTRACTThe evolution of peptide-specific CD4+T-cell responses to acute viral infections of humans is poorly understood. We analyzed the response to parvovirus B19 (B19), a ubiquitous and clinically significant pathogen with a compact and conserved genome. The magnitude and breadth of the CD4+T-cell response to the two B19 capsid proteins were investigated using a set of overlapping peptides and gamma interferon-specific enzyme-linked immunospot assays of peripheral blood mononuclear cells (PBMCs) from a cohort of acutely infected individuals who presented with acute arthropathy. These were compared to those for a cohort of B19-specific immunoglobulin M-negative (IgM−), IgG+remotely infected individuals. Both cohorts of individuals were found to make broad CD4+responses. However, while the responses following acute infection were detectable ex vivo, responses in remotely infected individuals were only detected after culture. One epitope (LASEESAFYVLEHSSFQLLG) was consistently targeted by both acutely (10/12) and remotely (6/7) infected individuals. This epitope was DRB1*1501 restricted, and a major histocompatibility complex peptide tetramer stained PBMCs from acutely infected individuals in the range of 0.003 to 0.042% of CD4+T cells. Tetramer-positive populations were initially CD62Llo; unlike the case for B19-specific CD8+T-cell responses, however, CD62L was reexpressed at later times, as responses remained stable or declined slowly. This first identification of B19 CD4+T-cell epitopes, including a key immunodominant peptide, provides the tools to investigate the breadth, frequency, and functions of cellular responses to this virus in a range of specific clinical settings and gives an important reference point for analysis of peptide-specific CD4+T cells during acute and persistent virus infections of humans.
Characterization of Human Metapneumovirus F Protein-Promoted Membrane Fusion: Critical Roles for Proteolytic Processing and Low pH
Authors: Schowalter Rachel M. , Smith Stacy E. , Dutch Rebecca Ellis 
Published: October 2006

ABSTRACT
Human metapneumovirus (HMPV) is a recently described human pathogen of the pneumovirus subfamily within the paramyxovirus family. HMPV infection is prevalent worldwide and is associated with severe respiratory disease, particularly in infants. The HMPV fusion protein (F) amino acid sequence contains features characteristic of other paramyxovirus F proteins, including a putative cleavage site and potential N-linked glycosylation sites. Propagation of HMPV in cell culture requires exogenous trypsin, which cleaves the F protein, and HMPV, like several other pneumoviruses, is infectious in the absence of its attachment protein (G). However, little is known about HMPV F-promoted fusion, since the HMPV glycoproteins have yet to be analyzed separately from the virus. Using syncytium and luciferase reporter gene fusion assays, we determined the basic requirements for HMPV F protein-promoted fusion in transiently transfected cells. Our data indicate that proteolytic cleavage of the F protein is a stringent requirement for fusion and that the HMPV G protein does not significantly enhance fusion. Unexpectedly, we also found that fusion can be detected only when transfected cells are treated with trypsin and exposed to low pH, indicating that this viral fusion protein may function in a manner unique among the paramyxoviruses. We also analyzed the F protein cleavage site and three potential N-linked glycosylation sites by mutagenesis. Mutations in the cleavage site designed to facilitate endogenous cleavage did so with low efficiency, and our data suggest that all three N-glycosylation sites are utilized and that each affects cleavage and fusion to various degrees.
Persistent Hepatitis C Virus Infection In Vitro: Coevolution of Virus andHost
Authors: Zhong Jin , Gastaminza Pablo , Chung Josan , Stamataki Zania , Isogawa Masanori , Cheng Guofeng , McKeating Jane A. , Chisari Francis V. 
Published: October 2006

ABSTRACT
The
virological and cellular consequences of persistent hepatitis C virus
(HCV) infection have been elusive due to the absence of the requisite
experimental systems. Here, we report the establishment and the
characteristics of persistent in vitro infection of human
hepatoma-derived cells by a recently described HCV genotype 2a
infectious molecular clone. Persistent in vitro infection was
characterized by the selection of viral variants that displayed
accelerated expansion kinetics, higher peak titers, and increased
buoyant densities. Sequencing analysis revealed the selection of a
single adaptive mutation in the HCV E2 envelope protein that was
largely responsible for the variant phenotype. In parallel, as the
virus became more aggressive, cells that were resistant to infection
emerged, displaying escape mechanisms operative at the level of viral
entry, HCV RNA replication, or both. Collectively, these results reveal
the existence of coevolutionary events during persistent HCV infection
that favor survival of both virus and
host.
Assembly of Highly Infectious Rotavirus Particles Recoated with Recombinant Outer Capsid Proteins
Authors: Trask Shane D. , Dormitzer Philip R. 
Published: October 2006

ABSTRACT

Assembly of the rotavirus outer capsid is the final step of a complex pathway. In vivo, the later steps include a maturational membrane penetration that is dependent on the scaffolding activity of a viral nonstructural protein. In vitro, simply adding the recombinant outer capsid proteins VP4 and VP7 to authentic double-layered rotavirus subviral particles (DLPs) in the presence of calcium and acidic pH increases infectivity by a factor of up to 10
7
, yielding particles as infectious as authentic purified virions. VP4 must be added before VP7 for high-level infectivity. Steep dependence of infectious recoating on VP4 concentration suggests that VP4-VP4 interactions, probably oligomerization, precede VP4 binding to particles. Trypsin sensitivity analysis identifies two populations of VP4 associated with recoated particles: properly mounted VP4 that can be specifically primed by trypsin, and nonspecifically associated VP4 that is degraded by trypsin. A full complement of properly assembled VP4 is not required for efficient infectivity. Minimal dependence of recoating on VP7 concentration suggests that VP7 binds DLPs with high affinity. The parameters for efficient recoating and the characterization of recoated particles suggest a model in which, after a relatively weak interaction between oligomeric VP4 and DLPs, VP7 binds the particles and locks VP4 in place. Recoating will allow the use of infectious modified rotavirus particles to explore rotavirus assembly and cell entry and could lead to practical applications in novel immunization strategies.
In Vitro Resistance to the Human Immunodeficiency Virus Type 1 Maturation Inhibitor PA-457 (Bevirimat)
Authors: Adamson Catherine S. , Ablan Sherimay D. , Boeras Ioana , Goila-Gaur Ritu , Soheilian Ferri , Nagashima Kunio , Li Feng , Salzwedel Karl , Sakalian Michael , Wild Carl T. , Freed Eric O. 
Published: October 2006

ABSTRACT

3-
O
-(3′,3′-dimethylsuccinyl)betulinic acid (PA-457 or bevirimat) potently inhibits human immunodeficiency virus type 1 (HIV-1) maturation by blocking a late step in the Gag processing pathway, specifically the cleavage of SP1 from the C terminus of capsid (CA). To gain insights into the mechanism(s) by which HIV-1 could evolve resistance to PA-457 and to evaluate the likelihood of such resistance arising in PA-457-treated patients, we sought to identify and characterize a broad spectrum of HIV-1 variants capable of conferring resistance to this compound. Numerous independent rounds of selection repeatedly identified six single-amino-acid substitutions that independently confer PA-457 resistance: three at or near the C terminus of CA (CA-H226Y, -L231F, and -L231M) and three at the first and third residues of SP1 (SP1-A1V, -A3T, and -A3V). We determined that mutations CA-H226Y, CA-L231F, CA-L231M, and SP1-A1V do not impose a significant replication defect on HIV-1 in culture. In contrast, mutations SP1-A3V and -A3T severely impaired virus replication and inhibited virion core condensation. The replication defect imposed by SP1-A3V was reversed by a second-site compensatory mutation in CA (CA-G225S). Intriguingly, high concentrations of PA-457 enhanced the maturation of SP1 residue 3 mutants. The different phenotypes associated with mutations that confer PA-457 resistance suggest the existence of multiple mechanisms by which HIV-1 can evolve resistance to this maturation inhibitor. These findings have implications for the ongoing development of PA-457 to treat HIV-1 infection in vivo.
hsp72, a Host Determinant of Measles Virus Neurovirulence
Authors: Carsillo Thomas , Traylor Zachary , Choi Changsun , Niewiesk Stefan , Oglesbee Michael 
Published: October 2006

ABSTRACTTransient hyperthermia such as that experienced during febrile episodes increases expression of the major inducible 70-kDa heat shock protein (hsp72). Despite the relevance of febrile episodes to viral pathogenesis and the multiple in vitro roles of heat shock proteins in viral replication and gene expression, the in vivo significance of virus-heat shock protein interactions is unknown. The present work determined the in vivo relationship between hsp72 levels and neurovirulence of an hsp72-responsive virus using the mouse model of measles virus (MV) encephalitis. Transgenic C57BL/6 mice were created to constitutively overexpress hsp72 in neurons, and these mice were inoculated intracranially with Edmonston MV (Ed MV) at 42 h of age. The mean viral RNA burden in brain was approximately 2 orders of magnitude higher in transgenic animals than in nontransgenic animals 2 to 4 weeks postinfection, and this increased burden was associated with a fivefold increase in mortality. Mice were also challenged with an Ed MV variant exhibiting an attenuated in vitro response to hsp72-dependent stimulation of viral transcription (Ed N-522D). This virus exhibited an attenuated neuropathogenicity in transgenic mice, where mortality and viral RNA burdens were not significantly different from nontransgenic mice infected with either Ed N-522D or parent Ed MV. Collectively, these results indicate that hsp72 levels can serve as a host determinant of viral neurovirulence in C57BL/6 mice, reflecting the direct influence of hsp72 on viral gene expression.