bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hantavirus GnT Elements Mediate TRAF3 Binding and Inhibit RIG-I/TBK1-Directed Beta Interferon Transcription by Blocking IRF3 Phosphorylation
Authors: Matthys Valery S. , Cimica Velasco , Dalrymple Nadine A. , Glennon Nicole B. , Bianco Chris , Mackow Erich R. 
Published: January 2014

ABSTRACT
Hantaviruses successfully replicate in primary human endothelial cells by restricting the early induction of beta interferon (IFN-β) and interferon-stimulated genes (ISGs). Gn proteins from NY-1V, ANDV, and TULV, but not PHV, harbor elements in their 142-residue cytoplasmic tails (GnTs) that inhibit RIG-I/MAVS/TBK1-TRAF3-directed IFN-β induction. Here, we define GnT interactions and residues required to inhibit TRAF3-TBK1-directed IFN-β induction and IRF3 phosphorylation. We observed that GnTs bind TRAF3 via residues within the TRAF-N domain (residues 392 to 415) and that binding is independent of the MAVS-interactive TRAF-C domain (residues 415 to 568). We determined that GnT binding to TRAF3 is mediated by C-terminal degrons within NY-1V or ANDV GnTs and that mutations that add degrons to TULV or PHV GnTs confer TRAF3 binding. Further analysis of GnT domains revealed that TRAF3 binding is a discrete GnT function, independent of IFN regulation, and that residues 15 to 42 from the NY-1V GnT C terminus are required for inhibiting TBK1-directed IFN-β transcription. Mutagenesis of the NY-1V GnT revealed that altering tyrosine 627 (Y627A/S/F) abolished GnT regulation of RIG-I/TBK1-directed IRF3 phosphorylation and transcriptional responses of ISRE, κB, and IFN-β promoters. Moreover, GnTs from NY-1V, ANDV, and TULV, but not PHV, inhibited RIG-I-directed IRF3 phosphorylation. Collectively, these findings suggest a novel role for GnTs in regulating RIG-I/TBK1 pathway-directed IRF3 phosphorylation and IFN-β induction and define virulence determinants within GnTs that may permit the attenuation of pathogenic hantaviruses.

IMPORTANCE
These findings provide a mechanism for selected hantavirus GnT interactions to regulate RIG-I/TBK1 signaling responses required for IFN-β induction by inhibiting TBK1 phosphorylation of IRF3. These studies culminate in showing that a single GnT residue, Y627, is required for the NY-1V GnT to inhibit RIG-I/TBK1-directed IRF3 phosphorylation and IFN-β induction. These findings define a potential virulence determinant within the NY-1V GnT that may permit hantavirus attenuation.
Deletion of the Vaccinia Virus N2L Gene Encoding an Inhibitor of IRF3 Improves the Immunogenicity of Modified Vaccinia Virus Ankara Expressing HIV-1 Antigens
Authors: García-Arriaza Juan , Gómez Carmen E. , Sorzano Carlos Óscar S. , Esteban Mariano 
Published: January 2014

ABSTRACT

A modified vaccinia virus Ankara poxvirus vector expressing the HIV-1 Env, Gag, Pol, and Nef antigens from clade B (MVA-B) is currently being tested in clinical trials. To improve its immunogenicity, we have generated and characterized the immune profile of MVA-B containing a deletion of the vaccinia viral gene
N2L
, which codes for an inhibitor of IRF3 (MVA-B ΔN2L). Deletion of
N2L
had no effect on virus growth kinetics or on the expression of HIV-1 antigens; hence, the N2 protein is not essential for MVA replication. The innate immune responses triggered by MVA-B ΔN2L revealed an increase in beta interferon, proinflammatory cytokines, and chemokines. Mouse prime-boost protocols showed that MVA-B ΔN2L improves the magnitude and polyfunctionality of HIV-1-specific CD4
+
and CD8
+
T cell adaptive and memory immune responses, with most of the HIV-1 responses mediated by CD8
+
T cells. In the memory phase, HIV-1-specific CD8
+
T cells with an effector phenotype were predominant and in a higher percentage with MVA-B ΔN2L than with MVA-B. In both immunization groups, CD4
+
and CD8
+
T cell responses were directed mainly against Env. Furthermore, MVA-B ΔN2L in the memory phase enhanced levels of antibody against Env. For the vector immune responses, MVA-B ΔN2L induced a greater magnitude and polyfunctionality of VACV-specific CD8
+
T memory cells than MVA-B, with an effector phenotype. These results revealed the immunomodulatory role of
N2L
, whose deletion enhanced the innate immunity and improved the magnitude and quality of HIV-1-specific T cell adaptive and memory immune responses. These findings are relevant for the optimization of poxvirus vectors as vaccines.

IMPORTANCE
On the basis of the limited efficacy of the RV144 phase III clinical trial, new optimized poxvirus vectors as vaccines against HIV/AIDS are needed. Here we have generated and characterized a new HIV/AIDS vaccine candidate on the basis of the poxvirus MVA vector expressing HIV-1 Env, Gag, Pol, and Nef antigens (MVA-B) and containing a deletion in the vaccinia virus
N2L
gene. Our findings revealed the immunomodulatory role of
N2L
and proved that its deletion from the MVA-B vector triggered an enhanced innate immune response in human macrophages and monocyte-derived dendritic cells. Furthermore, in immunized mice, MVA-B ΔN2L induced improvements in the magnitude and quality of adaptive and memory HIV-1-specific CD4
+
and CD8
+
T cell immune responses, together with an increase in the memory phase of levels of antibody against Env. Thus, the selective deletion of the
N2L
viral immunomodulatory gene is important for the optimization of MVA vectors as HIV-1 vaccines.
Dimerization of Flavivirus NS4B Protein
Authors: Zou Jing , Xie Xuping , Lee Le Tian , Chandrasekaran Ramya , Reynaud Aline , Yap Lijian , Wang Qing-Yin , Dong Hongping , Kang Congbao , Yuan Zhiming , Lescar Julien , Shi Pei-Yong 
Published: January 2014

ABSTRACT

Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood. Here we report a robust method to express and purify dengue virus (DENV) and West Nile virus NS4B proteins. The NS4B proteins were expressed in
Escherichia coli
, reconstituted in dodecyl maltoside (DDM) detergent micelles, and purified to >95% homogeneity. The recombinant NS4B proteins dimerized
in vitro
, as evidenced by gel filtration, chemical cross-linking, and multiangle light scattering experiments. The dimeric form of NS4B was also detected when the protein was expressed alone in cells as well as in cells infected with DENV type 2 (DENV-2). Mutagenesis analysis showed that the cytosolic loop (amino acids 129 to 165) and the C-terminal region (amino acids 166 to 248) are responsible for NS4B dimerization.
trans
-Complementation experiments showed that (i) two genome-length RNAs containing distinct NS4B lethal mutations could not
trans
-complement each other, (ii) the replication defect of NS4B mutant RNA could be restored in cells containing DENV-2 replicons, and (iii) expression of wild-type NS4B protein alone was not sufficient to restore the replication of the NS4B mutant RNA. Collectively, the results indicate that
trans
-complementation of a lethal NS4B mutant RNA requires wild-type NS4B presented from a replication complex.

IMPORTANCE
The reported expression and purification system has made it possible to study the biochemistry and structure of flavivirus NS4B proteins. The finding of flavivirus NS4B dimerization and the mapping of regions important for NS4B dimerization provide the possibility to inhibit viral replication through blocking NS4B dimerization. The requirement of NS4B in the context of the replication complex for successful
trans
-complementation enhances our understanding of NS4B in flavivirus replication.
Activity of Hexokinase Is Increased by Its Interaction with Hepatitis C Virus Protein NS5A
Authors: Ramière Christophe , Rodriguez Jonathan , Enache Liviu S. , Lotteau Vincent , André Patrice , Diaz Olivier 
Published: January 2014

ABSTRACT

The study of cellular central carbon metabolism modulations induced by viruses is an emerging field. Human cytomegalovirus (HCMV), herpes simplex virus (HSV), Kaposi's sarcoma-associated herpesvirus (KSHV), and hepatitis C virus (HCV) have been shown recently to reprogram cell metabolism to support their replication. During HCV infection the global glucidolipidic metabolism of hepatocytes is highly impacted. It was suggested that HCV might modify glucose uptake and glycolysis to increase fatty acids synthesis, but underlying mechanisms have not been completely elucidated. We thus investigated how HCV may modulate glycolysis. We observed that in infected Huh7.5 cells and in subgenomic replicon-positive Huh9.13 cells, glucose consumption as well as lactate secretion was increased. Using protein complementation assays and coimmunoprecipitation, we identified a direct interaction between the HCV NS5A protein and cellular hexokinase 2 (HK2), the first rate-limiting enzyme of glycolysis. NS5A expression was sufficient to enhance glucose consumption and lactate secretion in Huh7.5 cells. Moreover, determination of HK activity in cell homogenates revealed that addition of exogenous NS5A protein, either the full-length protein or its D2 or D3, but not D1, domain, was sufficient to increase enzyme activity. Finally, determination of recombinant HK2 catalytic parameters (
V
max
and

K
m

) in the presence of NS5A identified this viral protein as an activator of the enzyme. In summary, this study describes a direct interaction between HCV NS5A protein and cellular HK2 which is accompanied by an increase in HK2 activity that might contribute to an increased glycolysis rate during HCV infection.

IMPORTANCE
Substantial evidence indicates that viruses reprogram the central carbon metabolism of the cell to support their replication. Nevertheless, precise underlying mechanisms are poorly described. Metabolic pathways are structured as connected enzymatic cascades providing elemental biomolecular blocks necessary for cell life and viral replication. In this study, we observed an increase in glucose consumption and lactate secretion in HCV-infected cells, revealing higher glycolytic activity. We also identified an interaction between the HCV NS5A nonstructural protein and cellular hexokinase 2, the first rate-limiting enzyme of glycolysis. This interaction results in an enhancement of catalytic parameters of the enzyme, which might explain, at least in part, the aerobic glycolysis shift observed in HCV-infected cells.
T Cells Modulate Epstein-Barr Virus Latency Phenotypes during Infection of Humanized Mice
Authors: Heuts Frank , Rottenberg Martin E. , Salamon Daniel , Rasul Eahsan , Adori Monika , Klein George , Klein Eva , Nagy Noemi 
Published: January 2014

ABSTRACTHuman B cells, the main target of Epstein-Barr virus (EBV), can display several types of latent viral protein expression, denoted 0, I, IIa, IIb, or III. Of these, only type III expression induces proliferation of cellsin vitro. These latency types are present at specific stages of infection and are also characteristic of different tumor types, but their generation is not fully understood. In this study, we analyzed the role of T cells in the regulation of EBV viral latency by using humanized NOD/SCID/IL2Rγ−/−mice. Several spleens presented macroscopic tumors 4 weeks after infection. Explanted spleen B cells from some of the EBV-infected mice proliferatedin vitro, but this was usually lowered when cyclosporine was added to the cultures. This suggested that thein vitrogrowth of EBV-infected B cells required T cell help; thus, cells other than type III cells were also present in the spleens. Quantitative PCR analysis of promoter activities specific for the different EBV latency types confirmed that in addition to type III cells, type IIa and type I cells were present in the spleen. The relative usage of the viral promoter specific for I and IIa latency types (Q promoter) was higher in CD8+cell-depleted mice, and it was absent from CD4+cell-depleted mice. These results indicate that CD4+T cells are necessary for the generation/maintenance of cells with latency I/IIa in the humanized mice. CD4+T cells contributed to this process through their CD40L expression.IMPORTANCEAt primary infection with EBV, the infected B cells are proliferating and express viral proteins that have transforming potential. However, when the acute infection is resolved, in healthy individuals EBV is carried by a small fraction of B cells that express a restricted number of viral proteins unable to induce proliferation. Understanding the details of this transition is of fundamental importance. We studied this question in humanized mice by manipulating their different T cell compartments before and during infection with EBV. Our results indicate that CD4+T cells are responsible for the switch to a nonproliferating EBV program during primary infection with EBV.
Ecological and Genetic Determinants of Pepino Mosaic Virus Emergence
Authors: Moreno-Pérez Manuel G. , Pagán Israel , Aragón-Caballero Liliana , Cáceres Fátima , Fraile Aurora , García-Arenal Fernando 
Published: January 2014

ABSTRACT

Virus emergence is a complex phenomenon, which generally involves spread to a new host from a wild host, followed by adaptation to the new host. Although viruses account for the largest fraction of emerging crop pathogens, knowledge about their emergence is incomplete. We address here the question of whether
Pepino Mosaic Virus
(PepMV) emergence as a major tomato pathogen worldwide could have involved spread from wild to cultivated plant species and host adaptation. For this, we surveyed natural populations of wild tomatoes in southern Peru for PepMV infection. PepMV incidence, genetic variation, population structure, and accumulation in various hosts were analyzed. PepMV incidence in wild tomatoes was high, and a strain not yet reported in domestic tomato was characterized. This strain had a wide host range within the
Solanaceae
, multiplying efficiently in most assayed
Solanum
species and being adapted to wild tomato hosts. Conversely, PepMV isolates from tomato crops showed evidence of adaptation to domestic tomato, possibly traded against adaptation to wild tomatoes. Phylogenetic reconstructions indicated that the most probable ancestral sequence came from a wild
Solanum
species. A high incidence of PepMV in wild tomato relatives would favor virus spread to crops and its efficient multiplication in different
Solanum
species, including tomato, allowing its establishment as an epidemic pathogen. Later, adaptation to tomato, traded off against adaptation to other
Solanum
species, would isolate tomato populations from those in other hosts.

IMPORTANCE
Virus emergence is a complex phenomenon involving multiple ecological and genetic factors and is considered to involve three phases: virus encounter with the new host, virus adaptation to the new host, and changes in the epidemiological dynamics. We analyze here if this was the case in the recent emergence of
Pepino Mosaic Virus
(PepMV) in tomato crops worldwide. We characterized a new strain of PepMV infecting wild tomato populations in Peru. Comparison of this strain with PepMV isolates from tomato crops, plus phylogenetic reconstructions, supports a scenario in which PepMV would have spread to crops from wild tomato relatives, followed by adaptation to the new host and eventually leading to population isolation. Our data, which derive from the analysis of field isolates rather than from experimental evolution approaches, significantly contribute to understanding of plant virus emergence, which is necessary for its anticipation and prevention.
Rice Stripe Tenuivirus NSvc2 Glycoproteins Targeted to the Golgi Body by the N-Terminal Transmembrane Domain and Adjacent Cytosolic 24 Amino Acids via the COP I- and COP II-Dependent Secretion Pathway
Authors: Yao Min , Liu Xiaofan , Li Shuo , Xu Yi , Zhou Yijun , Zhou Xueping , Tao Xiaorong 
Published: January 2014

ABSTRACT

The NSvc2 glycoproteins encoded by
Rice stripe tenuivirus
(RSV) share many characteristics common to the glycoproteins found among
Bunyaviridae
. Within this viral family, glycoproteins targeting to the Golgi apparatus play a pivotal role in the maturation of the enveloped spherical particles. RSV particles, however, adopt a long filamentous morphology. Recently, RSV NSvc2 glycoproteins were shown to localize exclusively to the ER in Sf9 insect cells. Here, we demonstrate that the amino-terminal NSvc2 (NSvc2-N) targets to the Golgi apparatus in
Nicotiana benthamiana
cells, whereas the carboxyl-terminal NSvc2 (NSvc2-C) accumulates in the endoplasmic reticulum (ER). Upon coexpression, NSvc2-N redirects NSvc2-C from the ER to the Golgi bodies. The NSvc2 glycoproteins move together with the Golgi stacks along the ER/actin network. The targeting of the NSvc2 glycoproteins to the Golgi bodies was strictly dependent on functional anterograde traffic out of the ER to the Golgi bodies or on a retrograde transport route from the Golgi apparatus. The analysis of truncated and chimeric NSvc2 proteins demonstrates that the Golgi targeting signal comprises amino acids 269 to 315 of NSvc2-N, encompassing the transmembrane domain and 24 adjacent amino acids in the cytosolic tail. Our findings demonstrate for the first time that the glycoproteins from an unenveloped
Tenuivirus
could target Golgi bodies in plant cells.

IMPORTANCE
NSvc2 glycoprotein encoded by unenveloped
Rice stripe tenuivirus
(RSV) share many characteristics in common with glycoprotein found among
Bunyaviridae
in which all members have membrane-enveloped sphere particle. Recently, RSV NSvc2 glycoproteins were shown to localize exclusively to the ER in Sf9 insect cells. In this study, we demonstrated that the RSV glycoproteins could target Golgi bodies in plant cells. The targeting of NSvc2 glycoproteins to the Golgi bodies was dependent on active COP II or COP I. The Golgi targeting signal was mapped to the 23-amino-acid transmembrane domain and the adjacent 24 amino acids of the cytosolic tail of the NSvc2-N. In light of the evidence from viruses in
Bunyaviridae
that targeting Golgi bodies is important for the viral particle assembly and vector transmission, we propose that targeting of RSV glycoproteins into Golgi bodies in plant cells represents a physiologically relevant mechanism in the maturation of RSV particle complex for insect vector transmission.
The Majority of CD4
+
T-Cell Depletion during Acute Simian-Human Immunodeficiency Virus SHIV89.6P Infection Occurs in Uninfected Cells
Authors: Matrajt Laura , Younan Patrick M. , Kiem Hans-Peter , Schiffer Joshua T. 
Published: January 2014

ABSTRACT

Untreated human immunodeficiency virus (HIV) infection is characterized by depletion of CD4
+
T cells, ultimately leading to the impairment of host immune defenses and death. HIV-infected CD4
+
T cells die from direct virus-induced apoptosis and CD8 T-cell-mediated elimination, but a broader and more profound depletion occurs in uninfected CD4
+
T cells via multiple indirect effects of infection. We fit mathematical models to data from experiments that tested an HIV eradication strategy in which five macaques with a proportion of CD4
+
T cells resistant to simian-human immunodeficiency virus (SHIV) entry were challenged with SHIV89.6P, a highly pathogenic dual-tropic chimeric SIV-HIV viral strain that results in rapid loss of both SHIV-susceptible and SHIV-resistant CD4
+
T cells. Our results suggest that uninfected (bystander) cell death accounts for the majority of CD4
+
T-lymphocyte loss, with at least 60% and 99% of CD4
+
T cell death occurring in uninfected cells during acute and established infection, respectively. Mechanisms to limit the profound indirect killing effects associated with HIV infection may be associated with immune preservation and improved long-term survival.

IMPORTANCE
HIV infection induces a massive depletion of CD4
+
T cells, leading to profound immunodeficiency, opportunistic infections, and eventually death. While HIV induces apoptosis (programmed cell death) by directly entering and replicating in CD4
+
T cells, uninfected CD4
+
T cells also undergo apoptosis due to ongoing toxic inflammation in the region of infection. In this paper, we use mathematical models in conjunction with data from simian-human immunodeficiency virus SHIV89.6P infection in macaques (a model of HIV infection in humans) to estimate the percentage of cell death that occurs in uninfected cells during the initial period of infection. We reveal that the vast majority of cell death occurs in these cells, which are not infected. The “bystander effects” that lead to enormous reductions in the number of uninfected CD4
+
T cells may be a target for future interventions that aim to limit the extent of damage caused by HIV.
Viral Entry of Hepatitis B and D Viruses and Bile Salts Transportation Share Common Molecular Determinants on Sodium Taurocholate Cotransporting Polypeptide
Authors: Yan Huan , Peng Bo , Liu Yang , Xu Guangwei , He Wenhui , Ren Bijie , Jing Zhiyi , Sui Jianhua , Li Wenhui 
Published: January 2014

ABSTRACT
The liver bile acids transporter sodium taurocholate cotransporting polypeptide (NTCP) is responsible for the majority of sodium-dependent bile salts uptake by hepatocytes. NTCP also functions as a cellular receptor for viral entry of hepatitis B virus (HBV) and hepatitis D virus (HDV) through a specific interaction between NTCP and the pre-S1 domain of HBV large envelope protein. However, it remains unknown if these two functions of NTCP are independent or if they interfere with each other. Here we show that binding of the pre-S1 domain to human NTCP blocks taurocholate uptake by the receptor; conversely, some bile acid substrates of NTCP inhibit HBV and HDV entry. Mutations of NTCP residues critical for bile salts binding severely impair viral infection by HDV and HBV; to a lesser extent, the residues important for sodium binding also inhibit viral infection. The mutation S267F, corresponding to a single nucleotide polymorphism (SNP) found in about 9% of the East Asian population, renders NTCP without either taurocholate transporting activity or the ability to support HBV or HDV infection in cell culture. These results demonstrate that molecular determinants critical for HBV and HDV entry overlap with that for bile salts uptake by NTCP, indicating that viral infection may interfere with the normal function of NTCP, and bile acids and their derivatives hold the potential for further development into antiviral drugs.

IMPORTANCE
Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), are important human pathogens. Available therapeutics against HBV are limited, and there is no drug that is clinically available for HDV infection. A liver bile acids transporter (sodium taurocholate cotransporting polypeptide [NTCP]) critical for maintaining homeostasis of bile acids serves as a functional receptor for HBV and HDV. We report here that the NTCP-binding lipopeptide that originates from the first 47 amino acids of the pre-S1 domain of the HBV L protein blocks taurocholate transport. Some bile salts dose dependently inhibit HBV and HDV infection mediated by NTCP; molecular determinants of NTCP critical for HBV and HDV entry overlap with that for bile acids transport. This work advances our understanding of NTCP-mediated HBV and HDV infection in relation to NTCP's physiological function. Our results also suggest that bile acids or their derivatives hold potential for development into novel drugs against HBV and HDV infection.