bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetics of the Development of Protective Immunity in Mice Vaccinated with a Live Attenuated Retrovirus
Authors: Dittmer Ulf , Race Brent , Hasenkrug Kim J. 
Published: December 2019

ABSTRACT

Vaccination of mice with a live attenuated vaccine virus induces potent protection against subsequent challenge with pathogenic Friend retroviral complex. The kinetic studies presented here demonstrate protection from acute splenomegaly as early as 1 week postvaccination. At this time point virus-specific cytotoxic T lymphocytes (CTL) were demonstrable in direct chromium release assays. However, during the first 2 weeks after vaccination protection was incomplete since the mice were not protected against establishment of low-level persistent infections in the spleen. By 3 weeks postvaccination the animals were protected against the establishment of persistent virus as well as acute splenomegaly. The timing of this complete protection correlated with the presence of both virus-neutralizing antibodies and primed CTL in the immunized mice. Within 3 days of virus challenge, vaccinated mice showed high levels of activated B cells and CD4
+
and CD8
+
T cells, indicating an efficient priming of all lymphocyte subsets. Despite very limited replication of the vaccine virus, the protective effect was long lived and was still present 6 months after immunization.
Expression of Hepatitis C Virus Proteins Inhibits Signal Transduction through the Jak-STAT Pathway
Authors: Heim Markus H. , Moradpour Darius , Blum Hubert E. 
Published: December 2019

ABSTRACTHepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. Alpha interferon (IFN-α) therapy of chronic hepatitis C leads to a sustained response in 10 to 20% of patients only. The mechanisms of viral persistence and the pathogenesis of hepatitis C are poorly understood. We established continuous human cell lines, allowing the tightly regulated expression of the entire HCV open reading frame under the control of a tetracycline-responsive promoter. Using this in vitro system, we analyzed the effect of HCV proteins on IFN-induced intracellular signaling. Expression of HCV proteins in these cells strongly inhibited IFN-α-induced signal transduction through the Jak-STAT pathway. Inhibition occurred downstream of STAT tyrosine phosphorylation. Inhibition of the Jak-STAT pathway was not restricted to IFN-α-induced signaling but was observed in leukemia inhibitory factor-induced signaling through Stat3 as well. By contrast, tumor necrosis factor alpha-induced activation of the transcription factor NF-κB was not affected. Interference of HCV with IFN-α-induced signaling through the Jak-STAT pathway could contribute to the resistance to IFN-α therapy observed in the majority of patients and may represent a general escape strategy of HCV contributing to viral persistence and pathogenesis of chronic liver disease.
Transcription Originating in the Long Terminal Repeats of the Endogenous Mouse Mammary Tumor Virus MTV-3 Is Activated in Stat5a-Null Mice and Picks Up Hitchhiking Exons
Authors: Stegalkina Svetlana S. , Guerrero Annamaria , Walton Katherine D. , Liu Xiuwen , Robinson Gertraud W. , Hennighausen Lothar 
Published: December 2019

ABSTRACT
The enhancer within the long terminal repeats (LTRs) of acquired somatic mouse mammary tumor viruses (MMTV) can activate juxtaposed genes and induce mammary tumors. In contrast, germ line proviral MMTV genomes are integrated in the host genome and considered to be genetically confined transcription units. Here we demonstrate that transcription initiated in an MMTV provirus proceeds into flanking host sequences. We discovered multiple polyadenylated transcripts which are induced in Stat5a null mice. These range from 1.5 kb to more than 8 kb and are specifically expressed in mammary tissue from pregnant and lactating mice from the 129 but not C57BL/6 strain. The RNAs emanate from both LTRs of the endogenous MTV-3 provirus on chromosome 11 and proceed at least 10 kb into the juxtaposed genomic territory. Transcripts originating in the 5′ LTR splice from the native splice site within the MMTV envelope gene into at least six exons, three of which contain functional internal splice sites. The combination of alternative splicing and the use of several polyadenylation sites ensure the generation of multiple transcripts. To date no significant open reading frame has been discovered. Furthermore, we demonstrate that transcription from the MMTV 5′ LTR is highly active in the absence of Stat5a, a transcription factor that had been shown previously to be required for transcription from the MMTV LTR.
Involvement of the Cytoplasmic Domain of the Hemagglutinin-Neuraminidase Protein in Assembly of the Paramyxovirus Simian Virus 5
Authors: Schmitt Anthony P. , He Biao , Lamb Robert A. 
Published: December 2019

ABSTRACTEfficient assembly of enveloped viruses at the plasma membranes of virus-infected cells requires coordination between cytosolic viral components and viral integral membrane glycoproteins. As viral glycoprotein cytoplasmic domains may play a role in this coordination, we have investigated the importance of the hemagglutinin-neuraminidase (HN) protein cytoplasmic domain in the assembly of the nonsegmented negative-strand RNA paramyxovirus simian virus 5 (SV5). By using reverse genetics, recombinant viruses which contain HN with truncated cytoplasmic tails were generated. These viruses were shown to be replication impaired, as judged by small plaque size, reduced replication rate, and low maximum titers when compared to those features of wild-type (wt) SV5. Release of progeny virus particles from cells infected with HN cytoplasmic-tail-truncated viruses was inefficient compared to that of wt virus, but syncytium formation was enhanced. Furthermore, accumulation of viral proteins at presumptive budding sites on the plasma membranes of infected cells was prevented by HN cytoplasmic tail truncations. We interpret these data to indicate that formation of budding complexes, from which efficient release of SV5 particles can occur, depends on the presence of an HN cytoplasmic tail.
Efficacy of Antiretroviral Agents against Murine Replication-Competent Retrovirus Infection in Human Cells
Authors: Powell Sharon K. , Artlip Moria , Kaloss Michele , Brazinski Scott , Lyons Russette , McGarrity Gerard J. , Otto Edward 
Published: December 2019

ABSTRACT
Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone.
Poliovirus Mutants at Histidine 195 of VP2 Do Not Cleave VP0 into VP2 and VP4
Authors: Hindiyeh Musa , Li Qi-Han , Basavappa Ravi , Hogle James M. , Chow Marie 
Published: December 2019

ABSTRACT
The final stage of poliovirus assembly is characterized by a cleavage of the capsid precursor protein VP0 into VP2 and VP4. This cleavage is thought to be autocatalytic and dependent on RNA encapsidation. Analysis of the poliovirus empty capsid structure has led to a mechanistic model for VP0 cleavage involving a conserved histidine residue that is present in the surrounding environment of the VP0 cleavage site. Histidine 195 of VP2 (2195H) is hypothesized to activate local water molecules, thus initiating a nucleophilic attack at the scissile bond. To test this hypothesis, 2195H mutants were constructed and their phenotypes were characterized. Consistent with the requirement of VP0 cleavage for poliovirus infectivity, all 2195H mutants were nonviable upon introduction of the mutant genomes into HeLa cells. Replacement of 2195H with threonine or arginine resulted in the assembly of a highly unstable 150S virus particle. Further analyses showed that these particles contain genomic RNA and uncleaved VP0, criteria associated with the provirion assembly intermediate. These data support the involvement of 2195H in mediating VP0 cleavage during the final stages of virus assembly.
Establishment and Characterization of Cytopathogenic and Noncytopathogenic Pestivirus Replicons
Authors: Tautz N. , Harada T. , Kaiser A. , Rinck G. , Behrens S.-E. , Thiel H.-J. 
Published: December 2019

ABSTRACTDefective interfering particles (DIs) of bovine viral diarrhea virus (BVDV) have been identified and shown to be cytopathogenic (cp) in the presence of noncytopathogenic (noncp) helper virus. Moreover, a subgenomic (sg) RNA corresponding in its genome structure to one of those BVDV DIs (DI9) was replication competent in the absence of helper virus. We report here that an sg BVDV replicon which encodes from the viral proteins only the first three amino acids of the autoprotease Nproin addition to nonstructural (NS) proteins NS3 to NS5B replicates autonomously and also induces lysis of its host cells. This demonstrates that the presence of a helper virus is not required for the lysis of the host cell. On the basis of two infectious BVDV cDNA clones, namely, BVDV CP7 (cp) and CP7ins− (noncp), bicistronic replicons expressing proteins NS2-3 to NS5B were established. These replicons express, in addition to the viral proteins, the reporter gene encoding β-glucuronidase; the release of this enzyme from transfected culture cells was used to monitor cell lysis. Applying these tools, we were able to show that the replicon derived from CP7ins− does not induce cell lysis. Accordingly, neither Npronor any of the structural proteins are necessary to maintain the noncp phenotype. Furthermore, these sg RNAs represent the first pair of cp and noncp replicons which mimic complete BVDV CP7 and CP7ins− with respect to cytopathogenicity. These replicons will facilitate future studies aimed at the determination of the molecular basis for the cytopathogenicity of BVDV.
Effects of Targeting Herpes Simplex Virus Type 1 gD to the Endoplasmic Reticulum and
trans
-Golgi Network
Authors: Whiteley Alison , Bruun Birgitte , Minson Tony , Browne Helena 
Published: December 2019

ABSTRACT

Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) was modified to encode targeting signals known to localize proteins to either the endoplasmic reticulum (ER) or the
trans
-Golgi network. These motifs conferred the predicted targeting properties on gD in transfected cells as judged by immunofluorescence staining, and the exclusion of targeted gD from the cell surface was confirmed by the fact that these molecules exhibited substantially reduced activity in cell-cell fusion assays. Recombinant viruses expressing Golgi-targeted forms of gD grew to wild-type levels in noncomplementing cells, exhibited unaltered particle/infectivity ratios, and were found to contain wild-type levels of gD, whereas a recombinant expressing ER-retained gD was helper cell dependent and, when grown on noncomplementing cells, produced virions of low specific infectivity with greatly reduced levels of gD. These data imply that HSV-1 acquires its final membrane from a post-ER compartment and lend support to the view that the virus undergoes de-envelopment and reenvelopment steps during virus egress.
A Lentivirus Packaging System Based on Alternative RNA Transport Mechanisms To Express Helper and Gene Transfer Vector RNAs and Its Use To Study the Requirement of Accessory Proteins for Particle Formation and Gene Delivery
Authors: Srinivasakumar Narasimhachar , Schuening Friedrich G. 
Published: December 2019

ABSTRACT
A lentivirus-based packaging system was designed to reduce the chance of recombination between helper and gene transfer vector sequences by using the constitutive transport element (CTE) derived from Mason-Pfizer monkey virus for expression of the viral proteins and the Rev-Rev response element (RRE) combination for expression of the gene transfer vector. Using this approach, we evaluated a series of human immunodeficiency virus type 1 packaging constructs that express one or more accessory proteins (Vif, Vpr, and Vpu), in addition to the Gag and Pol proteins, for particle formation and virus stock production for gene transfer. Constructs that also express Vpr or both Vpr and Vpu produced more particles, as measured by a p24 assay, than did plasmids that did not contain these sequences. Transactivation experiments showed that the packaging plasmids that encode Vpr or both Vpr and Vpu also expressed a functional single-exon Tat protein. For these constructs, high-titer virus stocks could be prepared in the absence of a cotransfected Tat-expressing plasmid. Amphotropic-envelope-pseudotyped virus stocks prepared with all of the packaging constructs, irrespective of whether any of the accessory proteins were coexpressed, were equally efficient in transducing growth-arrested HeLa cells. The combination/mixed packaging system was compared to systems that were based on either the CTE alone or Rev and RRE for expression of both the packaging plasmid as well as the gene transfer vector. The combination/mixed packaging system was comparable to the other systems for production of virus stocks, suggesting that this design may prove to be safer for the eventual deployment of lentivirus vectors for therapeutic purposes.
Overexpression of Cyclin A Inhibits Augmentation of Recombinant Adeno-Associated Virus Transduction by the Adenovirus E4orf6 Protein
Authors: Grifman Mirta , Chen Nancie N. , Gao Guang-ping , Cathomen Toni , Wilson James M. , Weitzman Matthew D. 
Published: December 2019

ABSTRACT
The 34-kDa product of adenovirus E4 region open reading frame 6 (E4orf6) dramatically enhances transduction by recombinant adeno-associated virus vectors (rAAV). This is achieved by promoting the conversion of incoming single-stranded viral genomes into transcriptionally competent duplex molecules. The molecular mechanism for enhancing second-strand synthesis is not fully understood. In this study, we analyzed the cellular consequences of E4orf6 expression and the requirements for efficient rAAV transduction mediated by E4orf6. Expression of E4orf6 in 293 cells led to an inhibition of cell cycle progression and an accumulation of cells in S phase. This was preceded by specific degradation of cyclin A and p53, while the levels of other proteins involved in cell cycle control remained unchanged. In addition, the kinase activity of cdc2 was inhibited. We further showed that p53 expression is not necessary or inhibitory for augmentation of rAAV transduction by E4orf6. However, overexpression of cyclin A inhibited E4orf6-mediated enhancement of rAAV transduction. A cyclin A mutant incapable of recruiting protein substrates for cdk2 was unable to inhibit E4orf6-mediated augmentation. In addition, we created an E4orf6 mutant that is selectively defective in rAAV augmentation of transduction. Based on these findings, we suggest that cyclin A degradation represents a viral mechanism to disrupt cell cycle progression, resulting in enhanced viral transduction. Understanding the cellular pathways used during transduction will increase the utility of rAAV vectors in a wide range of gene therapy applications.
Adenoviruses Activate Human Dendritic Cells without Polarization toward a T-Helper Type 1-Inducing Subset
Authors: Rea Delphine , Schagen Frederik H. E. , Hoeben Rob C. , Mehtali Majid , Havenga Menzo J. E. , Toes Rene E. M. , Melief Cornelis J. M. , Offringa Rienk 
Published: December 2019

ABSTRACTHuman monocyte-derived dendritic cells (DC) infected with recombinant adenoviruses (rAd) are promising candidate vaccines for inducing protective immunity against pathogens and tumors. However, since some viruses are known to negatively affect DC function, it is important to investigate the interactions between rAd and DC. We now show that infection by rAd enhances the immunostimulatory capacity of immature human monocyte-derived DC through the upregulation of the costimulatory molecules CD80, CD86, and CD40 and the major histocompatibility complex class I and II molecules. Although rAd infection fails to induce the secretion of interleukin-12 (IL-12) and only marginally induces the expression of the DC maturation marker CD83, it acts in synergy with CD40 triggering in rendering DC fully mature. rAd-infected DC triggered through CD40 produce more IL-12 and are more efficient in eliciting T-helper type 1 responses than DC activated by CD40 triggering only. rAd lacking one or more of the early regions, E1, E2A, E3, and E4, which play an important role in virus-host cell interactions are equally capable of DC activation. Efficient DC infection requires a high multiplicity of infection (>1,000), a fact which can be attributed to the absence of the coxsackievirus and adenovirus receptor on this cell type. Despite the poor ability of DC to be infected by rAd, which may be improved by targeting rAd to alternative DC surface molecules, DC infected with all currently tested rAd constitute potent immunostimulators. Our study provides new insights into the interactions between two highly promising vaccine components, rAd and DC, and indicates that their combination into one vaccine may be very advantageous for the stimulation of T-cell immunity.
Use of a gp120 Binding Assay To Dissect the Requirements and Kinetics of Human Immunodeficiency Virus Fusion Events
Authors: Doranz Benjamin J. , Baik Sarah S. W. , Doms Robert W. 
Published: December 2019

ABSTRACT

Binding of the extracellular subunit of human immunodeficiency type 1 (HIV-1) envelope (Env) glycoprotein (gp120) to CD4 triggers the induction or exposure of a highly conserved coreceptor binding site in gp120 that helps mediate membrane fusion. Characterizing the structural features involved in gp120-coreceptor binding and the conditions under which binding occurs is important for understanding the fusion process, the evolution of pathogenic strains in vivo, the identification of novel anti-HIV compounds, and the development of HIV vaccines that utilize triggered structures of Env. Here we use the kinetics of interaction between CCR5 and gp120 to understand temporal and structural changes that occur during viral fusion. Using saturation binding and homologous competition analysis, we estimated the

K
d

of interaction between CCR5 and gp120 from the macrophage tropic HIV-1 strain JRFL to be 4 nM. Unlike Env-mediated fusion, gp120 binding to CCR5 did not require divalent cations or elevated temperatures. Binding was not significantly affected by the pH of binding, G-protein coupling of CCR5, or partial gp120 deglycosylation. Oligomeric, uncleaved JRFL gp140 failed to bind CCR5 despite its ability to bind CD4 and monoclonal antibody 17b, suggesting that the uncleaved ectodomain of gp41 interferes with full exposure of the chemokine receptor binding site. Exposure of the chemokine receptor binding site on gp120 could be induced rapidly by CD4, but exposure of this site was lost upon CD4 dissociation from gp120, indicating that the conformational changes in gp120 induced by CD4 binding are fully reversible. The functional gp120-soluble CD4 complex was remarkably stable over time and temperature ranges, offering the possibility that complexes in which the highly conserved coreceptor binding site in gp120 is exposed can be used for vaccine development.
Consistent Viral Evolutionary Changes Associated with the Progression of Human Immunodeficiency Virus Type 1 Infection
Authors: Shankarappa Raj , Margolick Joseph B. , Gange Stephen J. , Rodrigo Allen G. , Upchurch David , Farzadegan Homayoon , Gupta Phalguni , Rinaldo Charles R. , Learn Gerald H. , He Xi , Huang Xiao-Li , Mullins James I. 
Published: December 2019

ABSTRACTTo understand the high variability of the asymptomatic interval between primary human immunodeficiency virus type 1 (HIV-1) infection and the development of AIDS, we studied the evolution of the C2-V5 region of the HIV-1envgene and of T-cell subsets in nine men with a moderate or slow rate of disease progression. They were monitored from the time of seroconversion for a period of 6 to 12 years until the development of advanced disease in seven men. Based on the analysis of viral divergence from the founder strain, viral population diversity within sequential time points, and the outgrowth of viruses capable of utilizing the CXCR4 receptor (X4 viruses), the existence of three distinct phases within the asymptomatic interval is suggested: an early phase of variable duration during which linear increases (∼1% per year) in both divergence and diversity were observed; an intermediate phase lasting an average of 1.8 years, characterized by a continued increase in divergence but with stabilization or decline in diversity; and a late phase characterized by a slowdown or stabilization of divergence and continued stability or decline in diversity. X4 variants emerged around the time of the early- to intermediate-phase transition and then achieved peak representation and began a decline around the transition between the intermediate and late phases. The late-phase transition was also associated with failure of T-cell homeostasis (defined by a downward inflection in CD3+T cells) and decline of CD4+T cells to ≤200 cells/μl. The strength of these temporal associations between viral divergence and diversity, viral coreceptor specificity, and T-cell homeostasis and subset composition supports the concept that the phases described represent a consistent pattern of viral evolution during the course of HIV-1 infection in moderate progressors. Recognition of this pattern may help explain previous conflicting data on the relationship between viral evolution and disease progression and may provide a useful framework for evaluating immune damage and recovery in untreated and treated HIV-1 infections.