bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heterologous Human Immunodeficiency Virus Type 1 Lentiviral Vectors Packaging a Simian Immunodeficiency Virus-Derived Genome Display a Specific Postentry Transduction Defect in Dendritic Cells
Authors: Goujon Caroline , Jarrosson-Wuilleme Loraine , Bernaud Jeanine , Rigal Dominique , Darlix Jean-Luc , Cimarelli Andrea 
Published: August 2003

ABSTRACT

Heterologous lentiviral vectors (LVs) represent a way to address safety concerns in the field of gene therapy by decreasing the possibility of genetic recombination between vector and packaging constructs and the generation of replication-competent viruses. Using described LVs based on human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus MAC251 (SIV
MAC251
), we asked whether heterologous virion particles in which
trans
-acting factors belonged to HIV-1 and
cis
elements belonged to SIV
MAC251
(HIV-
siv
) would behave as parental homologous vectors in all cell types. To our surprise, we found that although the heterologous HIV-
siv
vector was as infectious as its homologous counterpart in most human cells, it was defective in the transduction of dendritic cells (DCs) and, to a lesser extent, macrophages. In DCs, the main postentry defect was observed in the formation of two-long-terminal-repeat circles, despite the fact that full-length proviral DNA was being synthesized and was associated with the nucleus. Taken together, our data suggest that heterologous HIV-
siv
vectors display a cell-dependent infectivity defect, most probably at a post-nuclear entry migration step. As homologous HIV and SIV vectors do transduce DCs, we believe that these results underscore the importance of a conserved interaction between
cis
elements and
trans
-acting viral factors that is lost or suboptimal in heterologous vectors and essential only in the transduction of certain cell types. For gene therapy purposes, these findings indicate that the cellular tropism of LVs can be modulated not only through the use of distinct envelope proteins or tissue-specific promoters but also through the specific combinatorial use of packaging and transfer vector constructs.
Rotavirus Infection Stimulates the Cl−Reabsorption Process across the Intestinal Brush-Border Membrane of Young Rabbits
Authors: Lorrot Mathie , Martin Sandra , Vasseur Monique 
Published: August 2003

ABSTRACTRotavirus is a major cause of infantile gastroenteritis worldwide. However, the mechanisms underlying fluid and electrolyte secretion associated with diarrhea remain largely unknown. We investigated the hypothesis that loss of Cl−into the luminal contents during rotavirus infection may be caused by a dysfunction in the chloride absorptive capacity across the intestinal brush-border membrane (BBM). The luminal Cl−concentrations in the entire small intestine of young rabbits infected with lapine rotavirus decreased at 1 and 2 days postinfection (dpi), indicating net Cl−absorption. At 7 dpi, luminal Cl−concentrations were slightly increased, indicating a moderate net Cl−secretion. By using a rapid filtration technique,36Cl uptake across BBM was quantified by modulating the alkali-metal ion, electrical, chloride, and/or proton gradients. Rotavirus infection caused an identical, 127% ± 24% increase in all Cl−uptake activities (Cl−/H+symport, Cl−conductance, and Cl−/anion exchange) observed across the intestinal BBM. The rotavirus activating effects on the symporter started at 1 dpi and persisted up to 7 dpi. Kinetic analyses revealed that rotavirus selectively affected the capacity parameter characterizing the symporter. We report the novel observation that rotavirus infection stimulated the Cl−reabsorption process across the intestinal BBM. We propose that the massive Cl−reabsorption in villi could partly overwhelm chloride secretion in crypt cells, which possibly increases during rotavirus diarrhea, the resulting imbalance leading to a moderate net chloride secretion.
Novel Recombinant Parapoxvirus Vectors Induce Protective Humoral and Cellular Immunity against Lethal Herpesvirus Challenge Infection in Mice
Authors: Fischer Timo , Planz Oliver , Stitz Lothar , Rziha Hanns-Joachim 
Published: August 2003

ABSTRACTOrf virus (ORFV;Parapoxvirus ovis) was used to develop a novel vector system for the generation of effective and safe live vaccines. Based on the attenuated ORFV strain D1701-V, recombinants were produced that express the glycoproteins gC (D1701-VrVgC) or gD (D1701-VrVgD) of the alphaherpesvirus of swine, pseudorabies virus (PRV). Expression of gC and gD was also demonstrated on the surface of recombinant virus-infected murine cells that do not produce infectious ORFV. Single or combined immunization with the ORFV recombinants protected different mouse strains of a host species nonpermissive for ORFV against a fulminant, lethal PRV challenge infection equal to immunization with PRV live vaccine. Most notably, even a single immunization with D1701-VrVgC was protective, whereas two applications of D1701-VrVgD were required for immune protection. The higher protective capacity of D1701-VrVgC correlated with the induction of a strong specific humoral immune response. This suggestion was supported by transfer experiments using sera from recombinant-immunized mice, which resulted in partial gC but not gD antibody-mediated protection of the naïve recipients. Remarkably, immunization of different immune-deficient mice demonstrated that the application of the PRV gC-expressing recombinant controlled the challenge infection in the absence of either CD4+or CD8+T cells, B cells, or an intact perforin pathway. In contrast, D1701-VrVgD-immunized mice lacking CD4+T cells exhibited reduced protection, whereas animals lacking CD8+T cells, B cells, or perforin resisted the challenge infection. The present study demonstrates the potential of these new vector vaccines to efficiently prime both protective humoral and cell-mediated immune mechanisms in a host species nonpermissive for the vector virus.
Transforming Growth Factor β1 Receptor II Is Downregulated by E1A in Adenovirus-Infected Cells
Authors: Tarakanova Vera L. , Wold William S. M. 
Published: August 2003

ABSTRACTTransforming growth factor β1 (TGF-β1) signaling is compromised in many tumors, thereby allowing the tumor to escape the growth-inhibitory and proapoptotic activities of the cytokine. Human adenoviruses interfere with a number of cellular pathways involved in cell cycle regulation and apoptosis, initially placing the cell in a “tumor-like” state by forcing quiescent cells into the cell cycle and also inhibiting apoptosis. We report that adenovirus-infected cells resemble tumor cells in that TGF-β1 signaling is inhibited. The levels of TGF-β1 receptor II (TβRII) in adenovirus-infected cells were decreased, and this decrease was mapped, by using virus mutants, to theE1Agene and to amino acids 2 to 36 and the C-terminal binding protein binding site in the E1A protein. The decrease in the TβRII protein was accompanied by a decrease in TβRII mRNA. The decrease in TβRII protein levels in adenovirus-infected cells was greater than the decrease in TβRII mRNA, suggesting that downregulation of the TβRII protein may occur through more than one mechanism. Surprisingly in this context, the half-lives of the TβRII protein in infected and uninfected cells were similar. TGF-β1 signaling was compromised in cells infected with wild-type adenovirus, as measured with 3TP-lux, a TGF-β-sensitive reporter plasmid expressing luciferase. Adenovirus mutants deficient in TβRII downregulation did not inhibit TGF-β1 signaling. TGF-β1 pretreatment reduced the relative abundance of adenovirus structural proteins in infected cells, an effect that was potentiated when cells were infected with mutants incapable of modulating the TGF-β signaling pathway. These results raise the possibility that inhibition of TGF-β signaling by E1A is a means by which adenovirus counters the antiviral defenses of the host.
Herpes Simplex Virus Type 2 Virion Host Shutoff Protein Regulates Alpha/Beta Interferon but Not Adaptive Immune Responses during Primary Infection In Vivo
Authors: Murphy Jenny A. , Duerst Rebecca J. , Smith Tracy J. , Morrison Lynda A. 
Published: August 2003

ABSTRACTThe herpes simplex virus (HSV) virion host shutoff (vhs) protein, the product of the UL41 (vhs) gene, is an important determinant of HSV virulence. vhs has been implicated in HSV interference with host antiviral immune responses, down-regulating expression of major histocompatibility complex molecules to help HSV evade host adaptive immunity. The severe attenuation of vhs-deficient viruses in vivo could reflect their inability to escape immune detection. To test this hypothesis, BALB/c or congenic SCID mice were infected intravaginally (i.vag.) with the HSV type 2 (HSV-2)vhsnull mutant 333d41 or thevhsrescue virus 333d41R. vhs-deficient virus remained severely attenuated in SCID mice compared with rescue virus, indicating that vhs regulation of adaptive immune responses does not influence HSV pathogenesis during acute infection. Innate antiviral effectors remain intact in SCID mice; prominent among these is alpha/beta interferon (IFN-α/β). The attenuation of HSV-2vhsmutants could reflect their failure to suppress IFN-α/β-mediated antiviral activity. To test this hypothesis, 129 and congenic IFN-α/β receptor-deficient (IFN-α/βR−/−) mice were infected i.vag. with wild-type virus,vhsnull mutants 333-vhsB or 333d41, or thevhsrescue virus 333d41R. Whereas vhs-deficient viruses showed greatly reduced replication in the genital mucosa of 129 mice compared with wild-type orvhsrescue viruses, they were restored to nearly wild-type levels of replication in IFN-α/βR−/−mice over the first 2 days postinfection. Only wild-type andvhsrescue viruses caused severe genital disease and hind limb paralysis in 129 mice, but infection of IFN-α/βR−/−mice restored the virulence of vhs-deficient viruses. vhs-deficient viruses replicated as vigorously as wild-type and rescue viruses in the nervous systems of IFN-α/βR−/−mice. Restoration was specific for thevhsmutation, because thymidine kinase-deficient HSV-2 did not regain virulence or the capacity to replicate in the nervous systems of IFN-α/βR−/−mice. Furthermore, the defect in the IFN-α/β response was required for restoration of vhs-deficient virus replication and virulence, but the IFN-α/β-stimulated protein kinase R pathway was not involved. Finally, vhs of HSV-2 has a unique capacity to interfere with the IFN-α/β response in vivo, because an HSV-1vhsnull mutant did not recover replication and virulence after i.vag. inoculation into IFN-α/βR−/−mice. These results indicate that vhs plays an important role early in HSV-2 pathogenesis in vivo by interfering with the IFN-α/β-mediated antiviral response.
Activation of Mitogen-Activated Protein Kinase and NF-κB Pathways by a Kaposi's Sarcoma-Associated Herpesvirus K15 Membrane Protein
Authors: Brinkmann Melanie M. , Glenn Mark , Rainbow Lucille , Kieser Arnd , Henke-Gendo Cornelia , Schulz Thomas F. 
Published: August 2003

ABSTRACT

The K15 gene of Kaposi's sarcoma-associated herpesvirus (also known as human herpesvirus 8) consists of eight alternatively spliced exons and has been predicted to encode membrane proteins with a variable number of transmembrane regions and a common C-terminal cytoplasmic domain with putative binding sites for SH2 and SH3 domains, as well as for tumor necrosis factor receptor-associated factors. These features are reminiscent of the latent membrane proteins LMP-1 and LMP2A of Epstein-Barr virus and, more distantly, of the STP, Tip, and Tio proteins of the related γ
2
-herpesviruses herpesvirus saimiri and herpesvirus ateles. These viral membrane proteins can activate a number of intracellular signaling pathways. We have therefore examined the abilities of different K15-encoded proteins to initiate intracellular signaling. We found that a 45-kDa K15 protein derived from all eight K15 exons and containing 12 predicted transmembrane domains in addition to the cytoplasmic domain activated the Ras/mitogen-activated protein kinase (MAPK) and NF-κB pathways, as well as (more weakly) the c-Jun N-terminal kinase/SAPK pathway. Activation of the MAPK and NF-κB pathways required phosphorylation of tyrosine residue 481 within a putative SH2-binding site (YEEVL). This motif was phosphorylated by the tyrosine kinases Src, Lck, Yes, Hck, and Fyn. The region containing the YEEVL motif interacted with tumor necrosis factor receptor-associated factor 2 (TRAF-2), and a dominant negative TRAF-2 mutant inhibited the K15-mediated activation of the Ras/MAPK pathway, suggesting the involvement of TRAF-2 in the initiation of these signaling routes. In contrast, several smaller K15 protein isoforms activated these pathways only weakly. All of the K15 isoforms tested were, however, localized in lipid rafts, suggesting that incorporation into lipid rafts is not sufficient to initiate signaling. Additional regions of K15, located presumably in exons 2 to 5, may therefore contribute to the activation of these pathways. These findings illustrate that the 45-kDa K15 protein engages pathways similar to LMP1, LMP2A, STP, Tip, and Tio but combines functional features that are separated between LMP1 and LMP2A or STP and Tip.
Interferon Regulatory Factor 7 Regulates Expression of Epstein-Barr Virus Latent Membrane Protein 1: a Regulatory Circuit
Authors: Ning Shunbin , Hahn Angela M. , Huye Leslie E. , Pagano Joseph S. 
Published: August 2003

ABSTRACT
We have shown previously that interferon regulatory factor 7 (IRF7), a multifunctional protein intimately involved in latent Epstein-Barr virus (EBV) infection, is induced as well as activated by EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein. Since the LMP1 promoter (LMP1p) contains an interferon-stimulated response element (ISRE), we hypothesized that IRF7 might be able to regulate LMP1 expression and thus participate in a regulatory circuit between these two genes. In this study, IRF7 was shown first to activate LMP1p in transient transfection assays. Compared with EBV nuclear antigen 2 (EBNA2), the most potent viral transactivator of LMP1p, IRF7 has a lesser effect (approximately 10% that of EBNA2) on induction of LMP1p. Study with IRF7 deletion mutants showed that IRF7 functional domains have similar effects on both the beta interferon (IFN-β) and LMP1 promoters in BJAB and 293 cells, and study with IRF7 phosphomimetic mutants showed that IRF7 phosphorylation may be involved in the activation of these two promoters. Further, the ISRE in LMP1p responds to IRF7 induction and IRF7 binds to this element. In the EBV-positive cell line P3HR1, which lacks the complete EBNA2 and EBV-encoded leader protein genes and hence expresses low-level LMP1, IRF7 alone can notably increase the endogenous LMP1 mRNA and protein levels. These results indicate that LMP1 is regulated by this host cell gene in addition to the viral factor, EBNA2, and may help to explain how LMP1 is expressed in type II latency in the absence of EBNA2. Moreover, IRF7 can regulate a viral gene in addition to a host cellular gene such as the IFN-β gene. Together with the previous data that LMP1 can induce IRF7 expression and facilitate IRF7 phosphorylation and nuclear translocation, these results suggest a positive regulatory circuit between IRF7 and LMP1.
Link between Genome Packaging and Rate of Budding for Rous Sarcoma Virus
Authors: Callahan Eric M. , Wills John W. 
Published: August 2003

ABSTRACT
The subcellular location at which genomic RNA is packaged by Gag proteins during retrovirus assembly remains unknown. Since the membrane-binding (M) domain is most critical for targeting Gag to the plasma membrane, changes to this determinant might alter the path taken through the cell and reduce the efficiency of genome packaging. In this report, a Rous sarcoma virus (RSV) mutant having two acidic-to-basic substitutions in the M domain is described. This mutant, designated Super M, produced particles much faster than the wild type, but the mutant virions were noninfectious and contained only 1/10 the amount of genomic RNA found in wild-type particles. To identify the cause(s) of these defects, we considered data that suggest that RSV Gag traffics through the nucleus to package the viral genome. Although inhibition of the CRM-1 pathway of nuclear export caused the accumulation of wild-type Gag in the nucleus, nuclear accumulation did not occur with Super M. The importance of the nucleocapsid (NC) domain in membrane targeting was also determined, and, importantly, deletion of the NC sequence prevented plasma membrane localization by wild-type Gag but not by Super M Gag. Based on these results, we reasoned that the enhanced membrane-targeting properties of Super M inhibit genome packaging. Consistent with this interpretation, substitutions that reestablished the wild-type number of basic and acidic residues in the Super M Gag M domain reduced the budding efficiency and restored genome packaging and infectivity. Therefore, these data suggest that Gag targeting and genome packaging are normally linked to ensure that RSV particles contain viral RNA.
Edmonston Measles Virus Prevents Increased Cell Surface Expression of Peptide-Loaded Major Histocompatibility Complex Class II Proteins in Human Peripheral Monocytes
Authors: Yilla Mamadi , Hickman Carole , McGrew Marcia , Meade Elizabeth , Bellini William J. 
Published: August 2003

ABSTRACTGamma interferon (IFN-γ) induces expression of the gene products of the major histocompatibility complex (MHC), whereas IFN-α/β can interfere with or suppress class II protein expression. In separate studies, measles virus (MV) was reported to induce IFN-α/β and to up-regulate MHC class II proteins. In an attempt to resolve this paradox, we examined the surface expression of MHC class I and class II proteins in MV-infected peripheral monocytes in the presence and absence of IFN-α/β. Infection of purified monocytes with Edmonston B MV resulted in an apparent increase in cell surface expression of HLA-A, -B, and -C class I proteins, but it had no effect on the expression of HLA-DR class II proteins. MV-infected purified monocytes expressed IFN-α/β, but no measurable IFN-γ expression was detected in supernatant fluids. Class II protein expression could be enhanced by coculture of purified monocytes with uninfected peripheral blood mononuclear cell (PBMC) supernatant. MV infection of PBMCs also did not affect expression of class II proteins, but the expression of HLA-A, -B, and -C class I proteins was increased two- to threefold in most donor cells. A direct role for IFN-α/β suppression of MHC class II protein expression was not evident in monocytes since MV suppressed class II protein expression in the absence of IFN-α/β. Taken together, these data suggest that MV interferes with the expression of peptide-loaded class II complexes, an effect that may potentially alter CD4+-T-cell proliferation and the cell-mediated immune responses that they help to regulate.
Variability at Human Immunodeficiency Virus Type 1 Subtype C Protease Cleavage Sites: an Indication of Viral Fitness?
Authors: de Oliveira Tulio , Engelbrecht Susan , Janse van Rensburg Estrelita , Gordon Michelle , Bishop Karen , zur Megede Jan , Barnett Susan W. , Cassol Sharon 
Published: August 2003

ABSTRACTNaturally occurring polymorphisms in the protease of human immunodeficiency virus type 1 (HIV-1) subtype C would be expected to lead to adaptive (compensatory) changes in protease cleavage sites. To test this hypothesis, we examined the prevalences and patterns of cleavage site polymorphisms in the Gag, Gag-Pol, and Nef cleavage sites of C compared to those in non-C subtypes. Codon-based maximum-likelihood methods were used to assess the natural selection and evolutionary history of individual cleavage sites. Seven cleavage sites (p17/p24, p24/p2, NC/p1, NC/TFP, PR/RT, RT/p66, and p66/IN) were well conserved over time and in all HIV-1 subtypes. One site (p1/p6gag) exhibited moderate variation, and four sites (p2/NC, TFP/p6pol, p6pol/PR, and Nef) were highly variable, both within and between subtypes. Three of the variable sites are known to be major determinants of polyprotein processing and virion production. P2/NC controls the rate and order of cleavage, p6gagis an important phosphoprotein required for virion release, and TFP/p6pol, a novel cleavage site in the transframe domain, influences the specificity of Gag-Pol processing and the activation of protease. Overall, 58.3% of the 12 HIV-1 cleavage sites were significantly more diverse in C than in B viruses. When analyzed as a single concatenated fragment of 360 bp, 96.0% of group M cleavage site sequences fell into subtype-specific phylogenetic clusters, suggesting that they coevolved with the virus. Natural variation at C cleavage sites may play an important role, not only in regulation of the viral cycle but also in disease progression and response to therapy.
The Moloney Murine Leukemia Virus Repressor Binding Site Represses Expression in Murine and Human Hematopoietic Stem Cells
Authors: Haas Dennis L. , Lutzko Carolyn , Logan Aaron C. , Cho Gerald J. , Skelton Dianne , Jin Yu Xiao , Pepper Karen A. , Kohn Donald B. 
Published: August 2003

ABSTRACT

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the
trans
-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34
+
CD38
−
hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.