bluuebunny/crossref_metadata_2025_split huggingface dataset:
Highly Efficient Induction of Protective Immunity by a Vaccinia Virus Vector Defective in Late Gene Expression
Authors: Holzer G. W. , Remp G. , Antoine G. , Pfleiderer M. , Enzersberger O. M. , Emsenhuber W. , Hämmerle T. , Gruber F. , Urban C. , Falkner F. G. , Dorner F. 
Published: December 2019

ABSTRACT

Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10
4
PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10
3
PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy.
Human Cytomegalovirus Infection of Caco-2 Cells Occurs at the Basolateral Membrane and Is Differentiation State Dependent
Authors: Jarvis Michael A. , Wang C. Edward , Meyers Heather L. , Smith Patricia P. , Corless Christopher L. , Henderson Gavin J. , Vieira Jeffrey , Britt William J. , Nelson Jay A. 
Published: December 2019

ABSTRACTEpithelial cells are known to be a major target for human cytomegalovirus (HCMV) infection; however, the analysis of virus-cell interactions has been difficult to approach due to the lack of in vitro models. In this study, we established a polarized epithelial cell model using a colon epithelial cell-derived cell line (Caco-2) that is susceptible to HCMV infection at early stages of cellular differentiation. Infection of polarized cells was restricted to the basolateral surface whereas virus was released apically, which was consistent with the apical and not basolateral surface localization of two essential viral glycoproteins, gB and gH. HCMV infection resulted in the development of a cytopathology characteristic of HCMV infection of colon epithelium in vivo, and infection did not spread from cell to cell. The inability of HCMV to infect Caco-2 cells at late stages of differentiation was due to a restriction at the level of viral entry and was consistent with the sequestration of a cellular receptor for HCMV. These observations provide the first evidence that restriction of HCMV replication in epithelial cells is due to a receptor-mediated phenomenon.
The Role of Human T-Lymphotropic Virus Type 1 (HTLV-1)-Infected Dendritic Cells in the Development of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis
Authors: Makino Masahiko , Shimokubo Satoshi , Wakamatsu Shin-Ichi , Izumo Shuji , Baba Masanori 
Published: December 2019

ABSTRACT

The development of human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is closely associated with the activation of T cells which are HTLV-1 specific but may cross-react with neural antigens (Ags). Immature dendritic cells (DCs), differentiated from normal donor monocytes by using recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin-4, were pulsed with HTLV-1 in vitro. The pulsed DCs contained HTLV-1 proviral DNA and expressed HTLV-1 Gag Ag on their surface 6 days after infection. The DCs matured by lipopolysaccharides stimulated autologous CD4
+
T cells and CD8
+
T cells in a viral dose-dependent manner. However, the proliferation level of CD4
+
T cells was five- to sixfold higher than that of CD8
+
T cells. In contrast to virus-infected DCs, DCs pulsed with heat-inactivated virions activated only CD4
+
T cells. To clarify the role of DCs in HAM/TSP development, monocytes from patients were cultured for 4 days in the presence of the cytokines. The expression of CD86 Ag on DCs was higher and that of CD1a Ag was more down-regulated than in DCs generated from normal monocytes. DCs from two of five patients expressed HTLV-1 Gag Ag. Furthermore, both CD4
+
and CD8
+
T cells from the patients were greatly stimulated by contact with autologous DCs pulsed with inactivated viral Ag as well as HTLV-1-infected DCs. These results suggest that DCs are susceptible to HTLV-1 infection and that their cognate interaction with T cells may contribute to the development of HAM/TSP.
Genetic Interaction of Flavivirus Nonstructural Proteins NS1 and NS4A as a Determinant of Replicase Function
Authors: Lindenbach Brett D. , Rice Charles M. 
Published: December 2019

ABSTRACT

Nonstructural protein 1 (NS1) of yellow fever virus (YF) is a glycoprotein localized to extracytoplasmic compartments within infected cells. We have previously shown that NS1 can be supplied in
trans
and is required for viral RNA replication, a process thought to occur in membrane-bound cytoplasmic complexes. Here we report that the NS1 gene from a related virus, dengue virus (DEN), is unable to function in the process of YF RNA replication. This virus-specific incompatibility leads to a lack of initial minus-strand accumulation, suggesting that DEN NS1 is unable to productively interact with the YF replicase. Based on a YF deletion mutant that requires NS1 in
trans
, a genetic screen for suppressor mutants was used to select virus variants able to utilize DEN NS1. In three independent selections, a single mutation was mapped to the NS4A gene, which encodes a putative transmembrane replicase component. This mutation, as well as several additional mutations, was engineered into the NS1-deficient genome and confirmed a genetic interaction between NS1 and NS4A. These findings suggest a potential mechanism for integrating NS1 into the cytoplasmic process of RNA replication.
Control of Cell Cycle Entry and Apoptosis in B Lymphocytes Infected by Epstein-Barr Virus
Authors: Spender Lindsay C. , Cannell Emma J. , Hollyoake Martine , Wensing Barbara , Gawn Jonathan M. , Brimmell Matthew , Packham Graham , Farrell Paul J. 
Published: December 2019

ABSTRACTInfection of human B cells with Epstein-Barr virus (EBV) results in activation of the cell cycle and cell growth. To interpret the mechanisms by which EBV activates the cell, we have assayed many proteins involved in control of the G0and G1phases of the cell cycle and regulation of apoptosis. In EBV infection most of the changes, including the early induction of cyclin D2, are dependent on expression of EBV genes, but an alteration in the E2F-4 profile was partly independent of viral gene expression, presumably occurring in response to signal transduction activated when the virus binds to its receptor, CD21. By comparing the expression of genes controlling apoptosis, including those encoding several members of the BCL-2 family of proteins, the known relative resistance of EBV-immortalized B-cell lines to apoptosis induced by low serum was found to correlate with expression of both BCL-2 and A20. A20 can be regulated by the NF-κB transcription factor, which is known to be activated by the EBV LMP-1 protein. Quantitative assays demonstrated a direct temporal relationship between LMP-1 protein levels and active NF-κB during the time course of infection.
Human Herpesvirus 8-Encoded Thymidine Kinase and Phosphotransferase Homologues Confer Sensitivity to Ganciclovir
Authors: Cannon Jennifer S. , Hamzeh Fayez , Moore Stacy , Nicholas John , Ambinder Richard F. 
Published: December 2019

ABSTRACT

Human herpesvirus 8 (HHV-8) sensitivity to the nucleoside analog ganciclovir (GCV) suggests the presence of a virally encoded kinase that catalyzes the initial phosphorylation of GCV. Analysis of the HHV-8 genome identified two candidate kinases: proteins encoded by open reading frame (ORF) 21, with homology to the herpesvirus thymidine kinases (TK), and ORF 36, with homology to the herpesvirus phosphotransferases (PT). Experiments presented here show that both ORF 21 and ORF 36 encode GCV kinase activities as demonstrated by GCV phosphorylation and GCV-mediated cell death. In both regards the PT homologue ORF 36 was more active than the TK homologue ORF 21. ORF 21, but not ORF 36, weakly sensitized cells to killing by penciclovir. Neither ORF sensitized cells to killing by (
E
)-5-(2-bromovinyl)-2′-deoxyuridine.
Characterization of a Chinese Hamster Ovary Cell Line Developed by Retroviral Insertional Mutagenesis That Is Resistant to Sindbis Virus Infection
Authors: Jan Jia-Tsrong , Byrnes Andrew P. , Griffin Diane E. 
Published: December 2019

ABSTRACTThe alphavirus Sindbis virus (SV) has a wide host range and infects many types of cultured cells in vitro. The outcome of infection is dependent on the strain of virus used for infection and the properties of the cells infected. To identify cellular determinants of susceptibility to SV infection we mutagenized Chinese hamster ovary (CHO) cells by retroviral insertion with a vector containing the neomycin resistance gene that allowed selection for integration into transcriptionally active genes. Cells were then selected for survival after infection with SV. The most resistant cell line (CHO-18.4m) exhibited delayed virus replication and virus-induced cell death, had a single retroviral insertion, and was defective in SV binding to the cell surface. Further analysis revealed that CHO-18.4m cells were deficient in the expression of the sulfated glycosaminoglycans heparan sulfate and chondroitin sulfate. This further confirms the importance of heparan sulfate as an attachment molecule for SV in vitro and demonstrates the usefulness of this technique for identifying cellular genes that are important for virus replication.
Highly Attenuated Vaccine Strains of Simian Immunodeficiency Virus Protect against Vaginal Challenge: Inverse Relationship of Degree of Protection with Level of Attenuation
Authors: Johnson R. Paul , Lifson Jeffrey D. , Czajak Susan C. , Cole Kelly Stefano , Manson Kelledy H. , Glickman Rhona , Yang Janet , Montefiori David C. , Montelaro Ronald , Wyand Michael S. , Desrosiers Ronald C. 
Published: December 2019

ABSTRACT
Three different deletion mutants of simian immunodeficiency virus (SIV) that vary in their levels of attenuation were tested for the ability to protect against mucosal challenge with pathogenic SIV. Four female rhesus monkeys were vaccinated by intravenous inoculation with SIVmac239Δ3, four with SIVmac239Δ3X, and four with SIVmac239Δ4. These three vaccine strains exhibit increasing levels of attenuation: Δ3 < Δ3X <Δ4. The vaccinated monkeys were challenged by vaginal exposure to uncloned, pathogenic SIVmac251 at 61 weeks after the time of vaccination. On the basis of viral RNA loads in plasma, cell-associated virus loads in peripheral blood, and CD4 cell counts, strong protective effects were observed in all three groups of vaccinated monkeys. However, the degree of protection correlated inversely with the level of attenuation; the least-attenuated strain, SIVmac239Δ3, gave the greatest protection. One monkey in the Δ3X group and two in the Δ4 group clearly became superinfected by the challenge virus, but these animals had levels of SIV RNA in plasma that were considerably lower than those of naive animals that were challenged in parallel. Protection against vaginal challenge appears easier to achieve than protection against intravenous challenge, since four other SIVmac239Δ4-vaccinated monkeys showed no protection when challenged intravenously with a much lower inoculum of the same challenge virus stock. Protection against vaginal challenge in the Δ4-vaccinated group occurred in the absence of detectable serum neutralizing activities and appeared to be associated with the development of an early SIV-specific cytotoxic-T-lymphocyte response. Our results demonstrate that mucosal protection can be achieved by systemic immunization with the highly attenuated SIVmac239Δ4 more than 1 year prior to the time of challenge.
Adenovirus-Mediated p21
(WAF1/SDII/CIP1)
Gene Transfer Induces Apoptosis of Human Cervical Cancer Cell Lines
Authors: Tsao Yeou-Ping , Huang Shyh-Jer , Chang Junn-Liang , Hsieh Jer-Tsong , Pong Rey-Chen , Chen Show-Li 
Published: December 2019

ABSTRACT

p21
(WAF1/SDII/CIP1)
(p21) arrests cell growth by inhibiting cyclin-depend kinases. To explore the potential of using p21 for the gene therapy of cervical cancer, we infected human papillomavirus (HPV)-positive cervical cancer cells (HeLa, SiHa, and Z172) and HPV-negative cervical cancer cells (C33A) with recombinant adenovirus encoding p21 cDNA. The results revealed that effective inhibition of cell growth could be achieved by sense p21 adenovirus but not antisense p21 adenovirus infection and occurred through apoptosis as measured by DNA fragmentation and chromatin condensation. Apoptosis was also observed in xenografts of human cervical cancer cells infected with sense p21 adenovirus, as confirmed by in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL). The apoptosis was not prevented by overexpression of the
bcl-2
transgene. To sum up, the apoptotic effect suggests that p21 should be a tumoricidal agent instead of a tumoristatic agent in preventing cervical cancers. In addition, our report substantiates the combination of the high efficiency of adenovirus vector-mediated gene delivery and the apoptotic effect of p21.
Minimum Requirements for Efficient Transduction of Dividing and Nondividing Cells by Feline Immunodeficiency Virus Vectors
Authors: Johnston Julie C. , Gasmi Mehdi , Lim Leland E. , Elder John H. , Yee Jiing-Kuan , Jolly Douglas J. , Campbell Kevin P. , Davidson Beverly L. , Sauter Sybille L. 
Published: December 2019

ABSTRACT

The development of gene delivery vectors based on feline immunodeficiency virus (FIV) is an attractive alternative to vectors based on primate sources for the delivery of genes into humans. To investigate the requirements for efficient transduction of dividing and nondividing cells by vector particles based on FIV, a series of packaging and vector constructs was generated for which viral gene expression was minimized and from which unnecessary
cis
-acting sequences were deleted. Pseudotyped vector particles produced in 293T cells were used to transduce various target cells, including contact-inhibited human skin fibroblasts and growth-arrested HT1080 cells. FIV vectors in which the U3 promoter was replaced with the cytomegalovirus promoter gave rise to over 50-fold-higher titers than FIV vectors containing the complete FIV 5′ long terminal repeat (LTR). Comparison of the transduction efficiencies of vectors containing different portions of the FIV Gag coding region indicates that at least a functional part of the FIV packaging signal (Ψ) is located within an area which includes the 5′ LTR and the first 350 bp of
gag
. Transduction efficiencies of vectors prepared without FIV
vif
and
orf2
accessory gene expression did not differ substantially from those of vectors prepared with accessory gene expression in either dividing or nondividing cells. The requirement for FIV
rev
-RRE was, however, demonstrated by the inefficient production of vector particles in the absence of
rev
expression. Together, these results demonstrate the efficient transduction of nondividing cells in vitro by a multiply attenuated FIV vector and contribute to an understanding of the minimum requirements for efficient vector production and infectivity. In addition, we describe the ability of an FIV vector to deliver genes in vivo into hamster muscle tissue.
The Carboxy-Terminal Acidic Domain of Rift Valley Fever Virus NSs Protein Is Essential for the Formation of Filamentous Structures but Not for the Nuclear Localization of the Protein
Authors: Yadani Fatima-Zahra , Kohl Alain , Préhaud Christophe , Billecocq Agnès , Bouloy Michèle 
Published: December 2019

ABSTRACT

The ambisense S segment of Rift Valley fever (RVF) virus (a phlebovirus in the
Bunyaviridae
family) codes for two proteins: the viral complementary-sense RNA for the N nucleoprotein and the genomic-sense RNA for the nonstructural protein NSs. Except for the fact that the NSs protein is phosphorylated and forms filamentous structures in the nuclei of infected cells (R. Swanepoel and N. K. Blackburn, J. Gen. Virol. 34:557–561, 1977), its role is poorly understood, especially since the replication cycle of all these viruses takes place in the cytoplasm. To investigate the mechanisms involved in filament formation, we expressed NSs in mammalian cells via a recombinant Semliki Forest virus and demonstrated that the protein alone was able to form structures similar to those observed in RVF virus-infected cells, indicating that the presence of other RVF virus proteins is not required for filament formation. The yeast two-hybrid system was used to show that the protein interacts with itself and to map the interacting domains. Various deletion and substitution mutants were constructed, and the mutant proteins were analyzed by immunoprecipitation, Western blotting and immunofluorescence. These experiments indicated that the 10 to 17 amino acids of the carboxy-terminal domain were involved in self-association of the protein and that deletion of this acidic carboxy-terminal domain prevents the protein from forming filaments but does not affect its nuclear localization. The role of two phosphorylation sites present in this domain was also investigated, but they were not found to have a major influence on the formation of the nuclear filament.
Broad-Spectrum Protection against Tombusviruses Elicited by Defective Interfering RNAs in Transgenic Plants
Authors: Rubio Teresa , Borja Marisé , Scholthof Herman B. , Feldstein Paul A. , Morris T. Jack , Jackson Andrew O. 
Published: December 2019

ABSTRACTWe have designed a DNA cassette to transcribe defective interfering (DI) RNAs of tomato bushy stunt virus (TBSV) and have investigated their potential to protect transgenicNicotiana benthamianaplants from tombusvirus infections. To produce RNAs with authentic 5′ and 3′ termini identical to those of the native B10 DI RNA, the DI RNA sequences were flanked by ribozymes (RzDI). When RzDI RNAs transcribed in vitro were mixed with parental TBSV transcripts and inoculated into protoplasts or plants, they became amplified, reduced the accumulation of the parental RNA, and mediated attenuation of the lethal syndrome characteristic of TBSV infections. Analysis of F1and F2RzDI transformants indicated that uninfected plants expressed the DI RNAs in low abundance, but these RNAs were amplified to very high levels during TBSV infection. By two weeks postinoculation with TBSV, all untransformedN. benthamianaplants and transformed negative controls died. Although infection of transgenic RzDI plants initially induced moderate to severe symptoms, these plants subsequently recovered, flowered, and set seed. Plants from the same transgenic lines also exhibited broad-spectrum protection against related tombusviruses but remained susceptible to a distantly related tombus-like virus and to unrelated viruses.
The Murine Gammaherpesvirus 68 v-Cyclin Gene Is an Oncogene That Promotes Cell Cycle Progression in Primary Lymphocytes
Authors: van Dyk Linda F. , Hess Jay L. , Katz Jonathan D. , Jacoby Meagan , Speck Samuel H. , Virgin Herbert W. 
Published: December 2019

ABSTRACTSeveral gammaherpesviruses contain open reading frames encoding proteins homologous to mammalian D-type cyclins. In this study, we analyzed the expression and function of the murine gammaherpesvirus 68 (γHV68) viral cyclin (v-cyclin). The γHV68 v-cyclin gene was expressed in lytically infected fibroblasts as a leaky-late mRNA of approximately 0.9 kb encoding a protein of approximately 25 kDa. To evaluate the effect of the γHV68 v-cyclin on cell cycle progression in primary lymphocytes and to determine if the γHV68 v-cyclin gene is an oncogene, we generated transgenic mice by using thelckproximal promoter to express the γHV68 v-cyclin in early T cells. Expression of the γHV68 v-cyclin significantly increased the number of thymocytes in cell culture, as determined by measuring both DNA content and incorporation of 5-bromo-2-deoxyuridine following in vivo pulse-labeling. Expression of the γHV68 v-cyclin interfered with normal thymocyte maturation, as shown by increased numbers of CD4+CD8+double-positive thymocytes and decreased numbers of CD4+or CD8+single-positive and T-cell-receptor-bright thymocytes and splenocytes in transgenic mice. Despite increased numbers of cycling thymocytes, γHV68–v-cyclin–transgenic mice did not have proportionately increased thymocyte numbers, and staining by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling demonstrated increased apoptosis in the thymi of v-cyclin-transgenic mice. Fifteen of 38 γHV68–v-cyclin–transgenic mice developed high-grade lymphoblastic lymphoma between 3 and 12 months of age. We conclude that (i) the γHV68 v-cyclin is expressed as a leaky-late gene in lytically infected cells, (ii) expression of the γHV68 v-cyclin in thymocytes promotes cell cycle progression and inhibits normal T-cell differentiation, and (iii) the γHV68 v-cyclin gene is an oncogene.
Colocalization of the Herpes Simplex Virus 1 U
L
4 Protein with Infected Cell Protein 22 in Small, Dense Nuclear Structures Formed prior to Onset of DNA Synthesis
Authors: Jahedi Sogol , Markovitz Nancy S. , Filatov Felix , Roizman Bernard 
Published: December 2019

ABSTRACT

Herpes simplex virus 1 infected cell protein 22 (ICP22) localizes in small, dense nuclear bodies of primate cells early in infection and in the more diffuse replicative complexes after the onset of DNA synthesis. U
L
4, a γ
2
protein, localizes in cytoplasm and in the small nuclear structures containing ICP22 but not in replicative complexes. In rabbit skin cells, both ICP22 and U
L
4 localize in the dense nuclear bodies late in infection. The results suggest that the small nuclear structures perform a function involving both proteins late in infection.
Alfalfa Mosaic Virus RNAs Serve as Cap Donors for Tomato Spotted Wilt Virus Transcription during Coinfection of
Nicotiana benthamiana
Authors: Duijsings Daniël , Kormelink Richard , Goldbach Rob 
Published: December 2019

ABSTRACT

Tomato spotted wilt virus (TSWV) was shown to use alfalfa mosaic virus (AMV) RNAs as cap donors in vivo during a mixed infection in
Nicotiana benthamiana
. By use of nested reverse transcription-PCR, TSWV N and NSs mRNAs provided with capped leader sequences derived from all four AMV RNAs could be cloned and sequenced. The sequence specificity of the putative TSWV endonuclease involved is discussed.