bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell-Type-Specific Gene Transfer into Human Cells with Retroviral Vectors That Display Single-Chain Antibodies
Authors: Jiang An , Chu Te-Hua T. , Nocken F. , Cichutek Klaus , Dornburg Ralph 
Published: December 2019

ABSTRACTThe successful application of human gene therapy protocols on a broad clinical basis will depend on the availability of in vivo cell-type-specific gene delivery systems. We have developed retroviral vector particles, derived from spleen necrosis virus (SNV), that display the antigen binding site of an antibody on the viral surface. Using retroviral vectors derived from SNV that displayed single-chain antibodies (scAs) directed against a carcinoembryonic antigen-cross-reacting cell surface protein, we have shown that an efficient, cell-type-specific gene delivery can be obtained. In this study, we tested whether other scAs displayed on SNV vector particles can also lead to cell-type-specific gene delivery. We displayed the following scAs on the retroviral surface: one directed against the human cell surface antigen Her2neu, which belongs to the epidermal growth factor receptor family; one directed against the stem cell-specific antigen CD34; and one directed against the transferrin receptor, which is expressed on liver cells and various other tissues. We show that retroviral vectors displaying these scAs are competent for infection in human cells which express the antigen recognized by the scA. Infectivity was cell type specific, and titers above 105CFU per ml of tissue culture supernatant medium were obtained. The density of the antigen on the target cell surface does not influence virus titers in vitro. Our data indicate that the SNV vector system is well suited for the development of a large variety of cell-type-specific targeting vectors.
Enhanced T-Cell Immunogenicity and Protective Efficacy of a Human Immunodeficiency Virus Type 1 Vaccine Regimen Consisting of Consecutive Priming with DNA and Boosting with Recombinant Fowlpox Virus
Authors: Kent Stephen J. , Zhao Anne , Best Susan J. , Chandler Jenalle D. , Boyle David B. , Ramshaw Ian A. 
Published: December 2019

ABSTRACTThe induction of human immunodeficiency virus (HIV)-specific T-cell responses is widely seen as critical to the development of effective immunity to HIV type 1 (HIV-1). Plasmid DNA and recombinant fowlpox virus (rFPV) vaccines are among the most promising safe HIV-1 vaccine candidates. However, the immunity induced by either vaccine alone may be insufficient to provide durable protection against HIV-1 infection. We evaluated a consecutive immunization strategy involving priming with DNA and boosting with rFPV vaccines encoding common HIV-1 antigens. In mice, this approach induced greater HIV-1-specific immunity than either vector alone and protected mice from challenge with a recombinant vaccinia virus expressing HIV-1 antigens. In macaques, a dramatic boosting effect on DNA vaccine-primed HIV-1-specific helper and cytotoxic T-lymphocyte responses, but a decline in HIV-1 antibody titers, was observed following rFPV immunization. The vaccine regimen protected macaques from an intravenous HIV-1 challenge, with the resistance most likely mediated by T-cell responses. These studies suggest a safe strategy for the enhanced generation of T-cell-mediated protective immunity to HIV-1.
Localization of a Baculovirus-Induced Chitinase in the Insect Cell Endoplasmic Reticulum
Authors: Thomas Carole J. , Brown Helen L. , Hawes Chris R. , Lee Bum Yong , Min Mi-Kyung , King Linda A. , Possee Robert D. 
Published: December 2019

ABSTRACT

Confocal immunofluorescence microscopy was used to demonstrate that the
Autographa californica
nucleopolyhedrovirus (Ac
M
NPV) chitinase was localized within the endoplasmic reticulum (ER) of virus-infected insect cells. This was consistent with removal of the signal peptide from the chitinase and an ER localization motif (KDEL) at the carboxyl end of the protein. Chitinase release from cells, a prerequisite for liquefaction of virus-infected insect larvae, appears to be aided by synthesis of the p10 protein. Deletion of
p10
from the Ac
M
NPV genome delayed the appearance of chitinase activity in the medium of virus-infected cells by 24 h and also delayed liquefaction of virus-infected
Trichoplusia ni
larvae by the same period.
Equine Infectious Anemia Virus Gag Polyprotein Late Domain Specifically Recruits Cellular AP-2 Adapter Protein Complexes during Virion Assembly
Authors: Puffer Bridget A. , Watkins S. C. , Montelaro Ronald C. 
Published: December 2019

ABSTRACT
We have identified an interaction between the equine infectious anemia virus (EIAV) late assembly domain and the cellular AP-2 clathrin-associated adapter protein complex. A YXXL motif within the EIAV Gag late assembly domain was previously characterized as a sequence critical for release of assembling virions. We now show that this YXXL sequence interacts in vitro with the AP-50 subunit of the AP-2 complex, while the functionally interchangeable late assembly domains carried by the Rous sarcoma virus p2b protein and human immunodeficiency virus type 1 p6 protein, which utilize PPPY and PTAPP L domains, respectively, do not bind AP-50 in vitro. In addition, EIAV late domain mutants containing mutations that have previously been shown to abrogate budding also exhibit marked decreases in AP-50 binding efficiencies. A role for AP-2 complex in viral assembly is supported by immunofluorescence analysis of EIAV-infected equine dermal cells demonstrating specific colocalization of the α adaptin subunit of AP-2 with the EIAV p9 protein at sites of virus budding on the plasma membrane. These data provide strong evidence that EIAV utilizes the cellular AP-2 complex to accomplish virion assembly and release.
Construction and Characterization of Hexon-Chimeric Adenoviruses: Specification of Adenovirus Serotype
Authors: Gall Jason G. D. , Crystal Ronald G. , Falck-Pedersen Erik 
Published: December 2019

ABSTRACTThis study has used the strategy of gene replacement to characterize the contribution of the adenovirus (Ad) capsid protein hexon to serotype definition. By replacing the Ad type 5 (Ad5) hexon gene with sequences from Ad2, we have changed the type specificity of the chimeric virus. The type-determining epitopes are primarily associated with loop 1 of hexon and, to a much lesser degree, with loop 2. In spite of the serotype distinctiveness of the chimeric hexon viruses, epitope similarity between the vectors resulted in a low level of cross-reactive neutralizing antibody, which in combination with activated cellular and innate arms of the immune system is sufficient to suppress gene transduction following readministration in vivo.
Antibody Neutralization-Resistant Primary Isolates of Human Immunodeficiency Virus Type 1
Authors: Parren Paul W. H. I. , Wang Meng , Trkola Alexandra , Binley James M. , Purtscher Martin , Katinger Hermann , Moore John P. , Burton Dennis R. 
Published: December 2019

ABSTRACTAlthough typical primary isolates of human immunodeficiency virus type 1 (HIV-1) are relatively neutralization resistant, three human monoclonal antibodies and a small number of HIV-1+human sera that neutralize the majority of isolates have been described. The monoclonal antibodies (2G12, 2F5, and b12) represent specificities that a putative vaccine should aim to elicit, since in vitro neutralization has been correlated with protection against primary viruses in animal models. Furthermore, a neutralization escape mutant to one of the antibodies (b12) selected in vitro remains sensitive to neutralization by the other two (2G12 and 2F5) (H. Mo, L. Stamatatos, J. E. Ip, C. F. Barbas, P. W. H. I. Parren, D. R. Burton, J. P. Moore, and D. D. Ho, J. Virol. 71:6869–6874, 1997), supporting the notion that eliciting a combination of such specificities would be particularly advantageous. Here, however, we describe a small subset of viruses, mostly pediatric, which show a high level of neutralization resistance to all three human monoclonal antibodies and to two broadly neutralizing sera. Such viruses threaten antibody-based antiviral strategies, and the basis for their resistance should be explored.
Resistance to Alpha/Beta Interferons Correlates with the Epizootic and Virulence Potential of Venezuelan Equine Encephalitis Viruses and Is Determined by the 5′ Noncoding Region and Glycoproteins
Authors: Spotts Deborah R. , Reich Robin M. , Kalkhan Mohammed A. , Kinney Richard M. , Roehrig John T. 
Published: December 2019

ABSTRACT
We compared the alpha/beta interferon (IFN-α/β) sensitivities of the TC-83 vaccine strain and 24 enzootic and epizootic Venezuelan equine encephalitis (VEE) isolates. The IFN-resistant or -sensitive phenotype correlated well with epizootic or enzootic potential. IFN-α/β resistance of Trinidad donkey (TRD) virus correlated with virulence determinants in the 5′ noncoding region and glycoproteins. Infection of mice lacking a functional IFN system with the IFN-sensitive TC-83 virus resulted in disease equivalent to that produced by the virulent, IFN-resistant TRD virus, further demonstrating that IFN resistance contributes to VEE virus virulence and is a biological marker of epizootic potential.
A Novel Simian Immunodeficiency Virus (SIVdrl)polSequence from the Drill Monkey,Mandrillus leucophaeus
Authors: Clewley J. P. , Lewis J. C. M. , Brown D. W. G. , Gadsby E. L. 
Published: December 2019

ABSTRACTThe drill monkey has been shown by serology and PCR to harbor a unique simian immunodeficiency virus (SIVdrl). Apolsequence, amplified from uncultured peripheral blood cells, is most closely related to the equivalent SIV sequences from the red-capped mangabey (SIVrcm), the sabaeus African green monkey (SIVagmSAB), and the chimpanzee (SIVcpz) and to the human immunodeficiency virus type 1 (HIV-1) sequence of humans. It is as yet unclear whether SIVdrl has a mosaic genome like SIVrcm and SIVagmSAB, is a member of the SIVcpz/HIV-1 lineage, or represents a novel primate lentivirus lineage.
Syncytium-Inhibiting Monoclonal Antibodies Produced against Human T-Cell Lymphotropic Virus Type 1-Infected Cells Recognize Class II Major Histocompatibility Complex Molecules and Block by Protein Crowding
Authors: Hildreth James E. K. 
Published: December 2019

ABSTRACTFour new monoclonal antibodies (MAbs) that inhibit human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation were produced by immunizing BALB/c mice with HTLV-1-infected MT2 cells. Immunoprecipitation studies and binding assays of transfected mouse cells showed that these MAbs recognize class II major histocompatibility complex (MHC) molecules. Previously produced anti-class II MHC antibodies also blocked HTLV-1-induced cell fusion. Coimmunoprecipitation and competitive MAb binding studies indicated that class II MHC molecules and HTLV-1 envelope glycoproteins are not associated in infected cells. Anti-MHC antibodies had no effect on human immunodeficiency virus type 1 (HIV-1) syncytium formation by cells coinfected with HIV-1 and HTLV-1, ruling out a generalized disruption of cell membrane function by the antibodies. High expression of MHC molecules suggested that steric effects of bound anti-MHC antibodies might explain their inhibition of HTLV-1 fusion. An anti-class I MHC antibody and a polyclonal antibody consisting of several nonblocking MAbs against other molecules bound to MT2 cells at levels similar to those of class II MHC antibodies, and they also blocked HTLV-1 syncytium formation. Dose-response experiments showed that inhibition of HTLV-1 syncytium formation correlated with levels of antibody bound to the surface of infected cells. The results show that HTLV-1 syncytium formation can be blocked by protein crowding or steric effects caused by large numbers of immunoglobulin molecules bound to the surface of infected cells and have implications for the structure of the cellular HTLV-1 receptor(s).
Cleavage of the Murine Leukemia Virus Transmembrane Env Protein by Human Immunodeficiency Virus Type 1 Protease: Transdominant Inhibition by Matrix Mutations
Authors: Kiernan Rosemary E. , Freed Eric O. 
Published: December 2019

ABSTRACT
We have identified mutations in the human immunodeficiency virus type 1 (HIV-1) matrix protein (MA) which block infectivity of virions pseudotyped with murine leukemia virus (MuLV) envelope (Env) glycoproteins without affecting infectivity conferred by HIV-1 Env or vesicular stomatitis virus G glycoproteins. This inhibition is very potent and displays a strong transdominant effect; infectivity is reduced more than 100-fold when wild-type and mutant molecular clones are cotransfected at a 1:1 ratio. This phenomenon is observed with both ecotropic and amphotropic MuLV Env. The MA mutations do not affect the incorporation of MuLV Env into virions. We demonstrate that in HIV-1 virions pseudotyped with MuLV Env, the HIV-1 protease (PR) efficiently catalyzes the cleavage of the p15(E) transmembrane (TM) protein to p12(E). Immunoprecipitation analysis of pseudotyped virions reveals that the mutant MA blocks this HIV-1 PR-mediated cleavage of MuLV TM. Furthermore, the transdominant inhibition exerted by the mutant MA on wild-type infectivity correlates with the relative level of p15(E) cleavage. Consistent with the hypothesis that abrogation of infectivity imposed by the mutant MA is due to inhibition of p15(E) cleavage, mutant virions are significantly more infectious when pseudotyped with a truncated p12(E) form of MuLV Env. These results indicate that HIV-1 Gag sequences can influence the viral PR-mediated processing of the MuLV TM Env protein p15(E). These findings have implications for the development of HIV-1-based retroviral vectors pseudotyped with MuLV Env, since p15(E) cleavage is essential for activating membrane fusion and virus infectivity.
Cell Cycle Progression in Monkey Cells Expressing Simian Virus 40 Small t Antigen from Adenovirus Vectors
Authors: Howe Alan K. , Gaillard Stéphanie , Bennett John S. , Rundell Kathleen 
Published: December 2019

ABSTRACT

The simian virus 40 small t antigen (small-t) is required for optimal viral replication and transformation, especially during the infection of nondividing cells, suggesting that the function of small-t is to promote cell cycle progression. The mechanism through which small-t promotes cell growth reflects, in part, its binding and inhibition of protein phosphatase 2A (PP2A). The use of recombinant adenoviruses allows small-t expression in a majority of cells in a population, thus providing a convenient source of cells for biochemical analyses. In monkey kidney CV1 cells, small-t expressed from these adenovirus vectors activated the mitogen-activated protein kinase (MAPK) pathway, induced JNK activity, and increased AP-1 DNA-binding activity, all in a PP2A-dependent manner. Expression of small-t also caused an increase in the phosphorylation of the Na
+
/H
+
antiporter, a mitogen-activated ion exchanger whose activity correlates with its phosphorylation. At least part of the antiporter phosphorylation induced by small-t reflected activation of the MAPK pathway, as suggested by results of assays using a chemical inhibitor of the MAPK-activating kinase, MEK. Finally, small-t expression from adenovirus vectors promoted efficient cell cycle progression by growth-arrested cells. These vectors should facilitate further analysis of effects of small-t on cell cycle mediators.
Human-Specific Integrations of the HERV-K Endogenous Retrovirus Family
Authors: Medstrand Patrik , Mager Dixie L. 
Published: December 2019

ABSTRACT
Several distinct families of endogenous retrovirus-like sequences (HERVs) exist in the genomes of humans and other primates. One of these families, the HERV-K group, contains members that encode functional proteins and that have been implicated in the etiology of insulin-dependent diabetes mellitus (IDDM). Because of potential functional and disease relevance, it is important to determine if there are HERV-K-associated genetic differences between individuals. In this study, we have investigated the divergence and evolutionary age of HERV-K long terminal repeats (LTRs). Thirty-seven LTRs, taken primarily from random human clones in GenBank, were aligned and grouped into nine clusters with decreasing sequence divergence. Cluster 1 sequences are 8.6% divergent, on average, whereas cluster 9 LTRs, represented by the LTRs of the fully sequenced HERV-K10 clone, show an average of only 1.1% divergence from each other. The evolutionary age of 18 LTRs from different clusters was then investigated by genomic PCR to determine presence or absence of the retroviral element in different primate species. LTRs from clusters of higher divergence were detected in monkeys and apes, whereas LTRs in clusters with lower divergence were acquired later in evolution. Notably, LTRs of cluster 9 were found only in humans at all nine loci examined. Genomic Southern analysis with an oligonucleotide probe specific for cluster 9 LTRs suggests that HERV-K elements with this type of LTR expanded independently in the genomes of humans and the great apes. This is the first report of endogenous retroviral integrations that are specific to humans and indicates that some HERVs have amplified much later than previously thought. These elements may still be actively transposing and may therefore represent a source of genetic variation linked to disease development.
Influence of Cell Polarity on Retrovirus-Mediated Gene Transfer to Differentiated Human Airway Epithelia
Authors: Wang Guoshun , Davidson Beverly L. , Melchert Paul , Slepushkin Vladimir A. , van Es Helmuth H. G. , Bodner Mordechai , Jolly Doug J. , McCray Paul B. 
Published: December 2019

ABSTRACTGene transfer with recombinant murine leukemia viruses (MuLV) provides the potential to permanently correct inherited lung diseases, such as cystic fibrosis (CF). Several problems prevent the application of MuLV-based recombinant retroviruses to lung gene therapy: (i) the lack of cell proliferation in mature pulmonary epithelia, (ii) inefficient gene transfer with a vector applied to the apical surface, and (iii) low titers of many retroviral preparations. We found that keratinocyte growth factor (KGF) stimulated proliferation of differentiated human tracheal and bronchial epithelia. Approximately 50% of epithelia divided in response to KGF as assessed by bromodeoxyuridine histochemistry. In airway epithelia stimulated to divide with KGF, high-titer ampho- and xenotropic enveloped vectors preferentially infected cells from the basal side. However, treatment with hypotonic shock or EGTA transiently increased transepithelial permeability, enhancing gene transfer with the vector applied to the mucosal surfaces of KGF-stimulated epithelia. Up to 35% of cells expressed the transgene after gene transfer. By using this approach, cells throughout the epithelial sheet, including basal cells, were targeted. Moreover, the Cl−transport defect in differentiated CF airway epithelia was corrected. These findings suggest that barriers to apical infection with MuLV can be overcome.
Adeno-Associated Virus Type 2-Mediated Gene Transfer: Role of Epidermal Growth Factor Receptor Protein Tyrosine Kinase in Transgene Expression
Authors: Mah Cathryn , Qing Keyun , Khuntirat Benjawan , Ponnazhagan Selvarangan , Wang Xu-Shan , Kube Dagmar M. , Yoder Mervin C. , Srivastava Arun 
Published: December 2019

ABSTRACT
Adeno-associated virus type 2 (AAV), a single-stranded, DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have recently documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays an important role in AAV-mediated transgene expression (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997) and that a strong correlation exists between the phosphorylation state of the ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing et al., J. Virol. 72:1593–1599, 1998). In this report, we document that treatment of cells with specific inhibitors of the epidermal growth factor receptor protein tyrosine kinase (EGF-R PTK) activity, such as tyrphostin, leads to significant augmentation of AAV transduction efficiency, and phosphorylation of the ssD-BP is mediated by the EGF-R PTK. Treatment of cells with EGF results in phosphorylation of the ssD-BP, whereas treatment with tyrphostin causes dephosphorylation of the ssD-BP and consequently leads to increased expression of the transgene. Furthermore, AAV transduction efficiency inversely correlates with expression of the EGF-R in different cell types, and stable transfection of the EGF-R cDNA causes phosphorylation of the ssD-BP, leading to significant inhibition in AAV-mediated transgene expression which can be overcome by the tyrphostin treatment. These data suggest that the PTK activity of the EGF-R is a crucial determinant in the life cycle of AAV and that further studies on the interaction between the EGF-R and the ssD-BP may yield new insights not only into its role in the host cell but also in the successful use of AAV vectors in human gene therapy.
Antiapoptotic but Not Antiviral Function of Human
bcl-2
Assists Establishment of Japanese Encephalitis Virus Persistence in Cultured Cells
Authors: Liao Ching-Len , Lin Yi-Ling , Shen Shih-Cheng , Shen Jing-Yih , Su Hong-Lin , Huang Yue-Ling , Ma Shiou-Hwa , Sun Yi-Ching , Chen Ko-Pei , Chen Li-Kuang 
Published: December 2019

ABSTRACT

Upon infection of Japanese encephalitis virus (JEV), baby hamster kidney (BHK-21) and Chinese hamster ovary (CHO) cells were killed by a mechanism involved in apoptosis. While readily established in a variety of cell lines, JEV persistence has never been successfully instituted in BHK-21 and CHO cells. Since stable expression of human
bcl-2
in BHK-21 cells has been shown to delay JEV-induced apoptosis, in this study we investigated whether JEV persistence could be established in such cells. When constitutively expressing
bcl-2
, but not its closest homolog,

bcl-X
L

, following a primary lytic infection, approximately 5 to 10% of BHK-21 and CHO cells became persistently JEV infected during a long-term culture. From the persistent bulks, several independent clones were selected and expanded to form stable cell lines that continuously produced infectious virus without marked cytopathic effects (CPE). Among these stable cell lines, the truncated nonstructural protein 1 (NS1) was also detected and was indistinguishable from the NS1 truncations previously observed in JEV-persistent murine neuroblastoma N18 cells. However, the stable expression of NS1 alone, regardless of whether it was truncated or full length, failed to render the engineered cells persistently infected by JEV, implying that aberrant NS1 proteins were likely a consequence of, rather than a cause for, the viral persistence. Enforced
bcl-2
expression, which did not affect virus replication and spread during the early phase of cytolytic infection, appeared to attain JEV persistence by restriction of virus-induced CPE. Our results suggest that it is the antiapoptotic, rather than the antiviral, effect of cellular
bcl-2
which plays a role in the establishment of JEV persistence.