bluuebunny/crossref_metadata_2025_split huggingface dataset:
Apical Gene Transfer into Quiescent Human and Canine Polarized Intestinal Epithelial Cells by Lentivirus Vectors
Authors: Seppen Jurgen , Barry Simon C. , Klinkspoor J. Henriette , Katen Louis J. , Lee Sum P. , Garcia J. Victor , Osborne William R. A. 
Published: July 2002

ABSTRACT
Intestinal epithelial cells secrete a protective luminal mucus barrier inhibiting viral gene transfer. Quiescent, polarized monolayers of primary epithelial cells from dog gallbladder and human colon are efficiently transduced through the apical mucus side by lentivirus vectors, suggesting their application to intestinal gene therapy.
Human Immunodeficiency Virus Type 1 Vif Protein Is an Integral Component of an mRNP Complex of Viral RNA and Could Be Involved in the Viral RNA Folding and Packaging Process
Authors: Zhang Hui , Pomerantz Roger J. , Dornadula Geethanjali , Sun Yong 
Published: July 2002

ABSTRACT

Virion infectivity factor (Vif) is a protein encoded by human immunodeficiency virus types 1 and 2 (HIV-1 and -2) and simian immunodeficiency virus, plus other lentiviruses, and is essential for viral replication either in vivo or in culture for nonpermissive cells such as peripheral blood lymphoid cells, macrophages, and H9 T cells. Defects in the
vif
gene affect virion morphology and reverse transcription but not the expression of viral components. It has been shown that Vif colocalizes with Gag in cells and Vif binds to the NCp7 domain of Gag in vitro. However, it seems that Vif is not specifically packaged into virions. The molecular mechanism(s) for Vif remains unknown. In this report, we demonstrate that HIV-1 Vif is an RNA-binding protein and specifically binds to HIV-1 genomic RNA in vitro. Further, Vif binds to HIV-1 RNA in the cytoplasm of virus-producing cells to form a 40S mRNP complex. Coimmunoprecipitation and in vivo UV cross-linking assays indicated that Vif directly interact with HIV-1 RNA in the virus-producing cells. Vif-RNA binding could be displaced by Gag-RNA binding, suggesting that Vif protein in the mRNP complex may mediate viral RNA interaction with HIV-1 Gag precursors. Furthermore, we have demonstrated that these Vif mutants that lose the RNA binding activity in vitro do not support
vif
-deficient HIV-1 replication in H9 T cells, suggesting that the RNA binding capacity of Vif is important for its function. Further studies regarding Vif-RNA interaction in virus-producing cells will be important for studying the function of Vif in the HIV-1 life cycle.
Direct Ex Vivo Kinetic and Phenotypic Analyses of CD8+T-Cell Responses Induced by DNA Immunization
Authors: Hassett Daniel E. , Slifka Mark K. , Zhang Jie , Whitton J. Lindsay 
Published: July 2002

ABSTRACTCD8+T-cell responses can be induced by DNA immunization, but little is known about the kinetics of these responses in vivo in the absence of restimulation or how soon protective immunity is conferred by a DNA vaccine. It is also unclear if CD8+T cells primed by DNA vaccines express the vigorous effector functions characteristic of cells primed by natural infection or by immunization with a recombinant live virus vaccine. To address these issues, we have used the sensitive technique of intracellular cytokine staining to carry out direct ex vivo kinetic and phenotypic analyses of antigen-specific CD8+T cells present in the spleens of mice at various times after (i) a single intramuscular administration of a plasmid expressing the nucleoprotein (NP) gene from lymphocytic choriomeningitis virus (LCMV), (ii) infection by a recombinant vaccinia virus carrying the same protein (vvNP), or (iii) LCMV infection. In addition, we have evaluated the rapidity with which protective immunity against both lethal and sublethal LCMV infections is achieved following DNA vaccination. The CD8+T-cell response in DNA-vaccinated mice was slightly delayed compared to LCMV or vvNP vaccinees, peaking at 15 days postimmunization. Interestingly, the percentage of antigen-specific CD8+T cells present in the spleen at day 15 and later time points was similar to that observed following vvNP infection. T cells primed by DNA vaccination or by infection exhibited similar cytokine expression profiles and had similar avidities for an immunodominant cytotoxic T lymphocyte epitope peptide, implying that the responses induced by DNA vaccination differ quantitatively but not qualitatively from those induced by live virus infection. Surprisingly, protection from both lethal and sublethal LCMV infections was conferred within 1 week of DNA vaccination, well before the peak of the CD8+T-cell response.
The 17 Nucleotides Downstream from the
env
Gene Stop Codon Are Important for Murine Leukemia Virus Packaging
Authors: Shin Yu Seung , Kim Jong-Mook , Kim Sunyoung 
Published: July 2002

ABSTRACT

We have identified a previously unknown nucleotide sequence important for the packaging of murine leukemia virus. This nucleotide sequence is located downstream from the stop codon of the
env
gene but does not overlap the polypurine tract. Deletion of 17 bp from this region resulted in a more than 10-fold decrease in viral titer. Consistent with this result, the deletion mutant showed a 20- to 30-fold drop in the amount of virion RNA in the culture supernatant. The total amount of virion protein in the culture supernatant was comparable for the deletion mutant and the parental virus, suggesting that the mutant construct could release the empty viral particles. These results suggested that the packaging signal sequence might be present at the two extreme sites of the viral genome, one in the region around the splice donor sequence downstream from the 5′ long terminal repeat (LTR) and the other immediately upstream from the 3′ LTR. Implications for gene therapy, especially in regard to construction of retroviral vectors and packaging constructs, are discussed.
Recombinant Respiratory Syncytial Virus That Does Not Express the NS1 or M2-2 Protein Is Highly Attenuated and Immunogenic in Chimpanzees
Authors: Teng Michael N. , Whitehead Stephen S. , Bermingham Alison , St. Claire Marisa , Elkins William R. , Murphy Brian R. , Collins Peter L. 
Published: July 2002

ABSTRACT

Mutant recombinant respiratory syncytial viruses (RSV) which cannot express the NS1 and M2-2 proteins, designated rA2ΔNS1 and rA2ΔM2-2, respectively, were evaluated as live-attenuated RSV vaccines. The rA2ΔNS1 virus contains a large deletion that should have the advantageous property of genetic stability during replication in vitro and in vivo. In vitro, rA2ΔNS1 replicated approximately 10-fold less well than wild-type recombinant RSV (rA2), while rA2ΔM2-2 had delayed growth kinetics but reached a final titer similar to that of rA2. Each virus was administered to the respiratory tracts of RSV-seronegative chimpanzees to assess replication, immunogenicity, and protective efficacy. The rA2ΔNS1 and rA2ΔM2-2 viruses were 2,200- to 55,000-fold restricted in replication in the upper and lower respiratory tracts but induced a level of RSV-neutralizing antibody in serum that was only slightly reduced compared to the level induced by wild-type RSV. The replication of wild-type RSV in immunized chimpanzees after challenge was reduced more than 10,000-fold at each site. Importantly, rA2ΔNS1 and rA2ΔM2-2 were 10-fold more restricted in replication in the upper respiratory tract than was the
cpts
248/404 virus, a vaccine candidate that retained mild reactogenicity in the upper respiratory tracts of 1-month-old infants. Thus, either rA2ΔNS1 or rA2ΔM2-2 might be appropriately attenuated for this age group, which is the major target population for an RSV vaccine. In addition, these results show that neither NS1 nor M2-2 is essential for RSV replication in vivo, although each is important for efficient replication.
Primary Structure of theHerpesvirus AtelesGenome
Authors: Albrecht Jens-Christian 
Published: July 2002

ABSTRACTHerpesvirus atelesis an agent indigenous to spider monkeys (Atelesspp.) and causes fulminant lymphomas in various New World primates. Structural and genetic relatedness led to the classification of this virus as a member of the genusRhadinovirus. It is most closely related toHerpesvirus saimiri. The 108,409-bp light DNA segment of the herpesvirus ateles strain 73 genome has two genes for U-RNA-like transcripts and 73 open reading frames, of which at least 6 show significant homologies to cellular genes (encoding complement control proteins, apoptosis-regulatory proteins, D-type cyclins, interleukin-8 receptors, and enzymes involved in nucleotide metabolism). The left terminal region of the light DNA segment bears the putative rhadinovirus oncogenetio.
Expression of the Totivirus
Helminthosporium victoriae 190S Virus
RNA-Dependent RNA Polymerase from Its Downstream Open Reading Frame in Dicistronic Constructs
Authors: Soldevila Ana I. , Ghabrial Said A. 
Published: July 2002

ABSTRACT

The undivided double-stranded RNA (dsRNA) genome of
Helminthosporium victoriae 190S virus
(Hv190SV) (genus
Totivirus
) consists of two large overlapping open reading frames (ORFs). The 5′-proximal ORF encodes a capsid protein (CP), and the downstream, 3′-proximal ORF encodes an RNA-dependent RNA polymerase (RDRP). Unlike the RDRPs of some other totiviruses, which are expressed as a CP-RDRP (Gag-Pol-like) fusion protein, the Hv190SV RDRP is detected only as a separate, nonfused polypeptide. In this study, we examined the expression of the RDRP ORF fused in frame to the coding sequence of the green fluorescent protein (GFP) in bacteria and
Schizosaccharomyces pombe
cells. The GFP fusions were readily detected in bacteria transformed with the monocistronic construct RDRP:GFP; expression of the downstream RDRP:GFP from the dicistronic construct CP-RDRP:GFP could not be detected. However, fluorescence microscopy and Western blot analysis indicated that RDRP:GFP was expressed at low levels from its downstream ORF in the dicistronic construct in
S. pombe
cells. No evidence that the RDRP ORF was expressed from a transcript shorter than the full-length dicistronic mRNA was found. A coupled termination-reinitiation mechanism that requires host or eukaryotic cell factors is proposed for the expression of Hv190SV RDRP.
A Group of V3 Sequences from Human Immunodeficiency Virus Type 1 Subtype E Non-Syncytium-Inducing, CCR5-Using Variants Are Resistant to Positive Selection Pressure
Authors: Shiino Teiichiro , Kato Kayoko , Kodaka Noriko , Miyakuni Tuyoshi , Takebe Yutaka , Sato Hironori 
Published: July 2002

ABSTRACTIn a human immunodeficiency virus type 1 (HIV-1)-infected individual, immune-pressure-mediated positive selection operates to maintain the antigenic polymorphism on the gp120 third variable (V3) loop. Recently, we suggested on the basis of sequencing C2/V3 segments from an HIV-1 subtype E-infected family that a V3 sequence lineage group of the non-syncytium-inducing (NSI) variants (group 1) was relatively resistant to positive selection pressure (35). To better understand the relationship between the intensity of positive selection pressure and cell tropism of the virus, we determined the linkage between each V3 genotype and its function of directing coreceptor preference and MT2 cell tropism. The biological characterization of a panel of V3 recombinant viruses showed that all of the group 1 V3 sequences could confer an NSI/CCR5-using (NSI/R5) phenotype on HIV-1LAI, whereas the group 2 V3 sequence, which was more positively charged than the group 1 sequence, dictated mainly a syncytium-inducing, CXCR4-using (SI/X4) phenotype. Phylogenetic analysis of C2/V3 sequences encoding group 1 or 2 V3 suggested that the variants carrying group 1 V3 are the ancestors of the intrafamilial infection and persisted in the family, while the variants carrying group 2 V3 evolved convergently from the group 1 V3 variants during disease progression in the individuals. Finally, a statistical test showed that the V3 sequence that could dictate an NSI/R5 phenotype had a synonymous substitution rate significantly higher than the nonsynonymous substitution rate. These data suggest that V3 sequences of the subtype E NSI/R5 variants are more resistant to positive selection pressure than those of the SI/X4 variants.
Epstein-Barr Virus LMP2A-Induced B-Cell Survival in Two Unique Classes of EμLMP2A Transgenic Mice
Authors: Caldwell Robert G. , Brown R. Clark , Longnecker Richard 
Published: July 2002

ABSTRACTLatent membrane protein 2A (LMP2A) is one of only two viral proteins expressed during latent Epstein-Barr virus (EBV) infections in human peripheral B cells. LMP2A blocks B-cell receptor (BCR) signal transduction in vitro by modulation of the Syk and Lyn protein tyrosine kinases. Five genetically unique LMP2A transgenic mouse lines (EμLMP2A) with B-cell lineage expression of LMP2A were generated in this study to analyze the importance of LMP2A expression in vivo. These animals can be grouped into EμLMP2ABCR+(TgB, Tg6, and TgC) and EμLMP2ABCR−(Tg7 and TgE) lines based on B-cell phenotype. LMP2A expression in bone marrow cells of EμLMP2ABCR−lines was associated with a bypass of normal B-lymphocyte developmental checkpoints inasmuch as immunoglobulin light-chain gene rearrangement occurred in the absence of complete immunoglobulin heavy-chain gene rearrangement. The resulting BCR-negative B cells were able to exit the bone marrow and colonize peripheral lymphoid organs. LMP2A expression in EμLMP2ABCR+lines was not associated with altered B-cell development in a genetically wild-type background. When crossed into a recombinase activating null (RAG−/−) genetic background, LMP2A expression in either RAG−/−EμLMP2ABCR+or RAG−/−EμLMP2ABCR−animals was able to provide a survival signal to BCR-negative splenic B cells. Additionally, bone marrow cells from all EμLMP2A animals were able to proliferate in response to interleukin-7-dependent developmental signals in vitro. These studies illustrate that LMP2A can provide a survival signal to BCR-negative B cells in two different groups of EμLMP2A transgenic mice.
Characterization of the
trans
-Activation Properties of Equine Herpesvirus 1 EICP0 Protein
Authors: Bowles Dawn E. , Kim Seong K. , O'Callaghan Dennis J. 
Published: July 2002

ABSTRACT

The EICP0 protein of equine herpesvirus 1 (EHV-1) is an early, viral regulatory protein that independently
trans
-activates EHV-1 immediate-early (IE), early, γ1 late, and γ2 late promoters. To assess whether this powerful
trans
-activator functions in conjunction with three other EHV-1 regulatory proteins to activate expression of the various classes of viral promoters, transient cotransfection assays were performed in which effector plasmids expressing the EICP22, EICP27, and IE proteins were used either singly or in combination with an EICP0 effector construct. These analyses revealed that (i) independently, the EICP0 and IE proteins are powerful
trans
-activators but do not function synergistically, (ii) the IE protein inhibits the ability of the EICP0 protein to
trans
-activate the IE, γ1 late, and γ2 late promoters, (iii) the EICP22 and EICP0 proteins do not function together to significantly
trans
-activate any EHV-1 promoter, and (iv) the EICP27 and EICP0 proteins function synergistically to
trans
-activate the early and γ1 late promoters. A panel of EICP0 truncation and deletion mutant plasmids was generated and used in experiments to define the domains of the 419-amino-acid (aa) EICP0 protein that are important for the
trans
-activation of each class of EHV-1 promoters. These studies revealed that (i) carboxy-terminal truncation mutants of the EICP0 protein exhibited a progressive loss of
trans
-activating ability as increasing portions of the carboxy terminus were removed, (ii) the amino terminus of the EICP0 protein containing the RING finger (aa 8 to 46) and the acidic region (aa 71 to 84) was necessary but not sufficient for activation of all classes of EHV-1 promoters, (iii) the RING finger was absolutely essential for activation of EHV-1 promoters, since deletion of the entire RING finger motif (aa 8 to 46) or a portion of it (aa 19 to 30) completely abrogated the ability of these mutants to activate any promoter tested, (iv) the acidic region contributed to the ability of the EICP0 protein to activate the early and γ1 late promoters, and deletion of the acidic region enhanced the ability of this mutant to activate the IE promoter, (v) the carboxy terminus (aa 325 to 419), which is rich in glutamine residues, was dispensable for the EICP0
trans
-activation function, (vi) a motif resembling a nuclear localization signal (aa 289 to 293) was unnecessary for the EICP0 protein to
trans
-activate promoters of any temporal class, and (vii) the EICP0 protein was phosphorylated during infection, and deletion of the serine-rich region (aa 210 to 217), a potential site for phosphorylation, reduced by more than 70% the ability of the EICP0 protein to activate the γ2 late class of promoters.
Molecular Tectonic Model of Virus Structural Transitions: the Putative Cell Entry States of Poliovirus
Authors: Belnap David M. , Filman David J. , Trus Benes L. , Cheng Naiqian , Booy Frank P. , Conway James F. , Curry Stephen , Hiremath Chaitanya N. , Tsang Simon K. , Steven Alasdair C. , Hogle James M. 
Published: July 2002

ABSTRACT
Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at ∼22-Å resolution by cryo-electron microscopy. The 135S and 80S particles are both ∼4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 Å were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell.
The Porcine Humoral Immune Response against Pseudorabies Virus Specifically Targets Attachment Sites on Glycoprotein gC
Authors: Ober Bertram T. , Teufel Berthold , Wiesmüller Karl-Heinz , Jung Günther , Pfaff Eberhard , Saalmüller Armin , Rziha Hanns-Joachim 
Published: July 2002

ABSTRACT

High titers of virus-neutralizing antibodies directed against glycoprotein gC of
Pseudorabies virus
(PRV) (
Suid herpesvirus 1
) are generally observed in the serum of immunized pigs. A known function of the glycoprotein gC is to mediate attachment of PRV to target cells through distinct viral heparin-binding domains (HBDs). Therefore, it was suggested that the virus-neutralizing activity of anti-PRV sera is directed against HBDs on gC. To address this issue, sera with high virus-neutralizing activity against gC were used to characterize the anti-gC response. Epitope mapping demonstrated that amino acids of HBDs are part of an antigenic antibody binding domain which is located in the N-terminal part of gC. Binding of antibodies to this antigenic domain of gC was further shown to interfere with the viral attachment. Therefore, these results show that the viral HBDs are accessible targets for the humoral anti-PRV response even after tolerance induction against self-proteins, which utilize similar HBDs to promote host protein-protein interactions. The findings indicate that the host's immune system can specifically block the attachment function of PRV gC. Since HBDs promote the attachment of a number of herpesviruses, the design of future antiherpesvirus vaccines should aim to induce a humoral immune response that prevents HBD-mediated viral attachment.
The Putative Natural Killer Decoy Early Genem04(gp34) of Murine Cytomegalovirus Encodes an Antigenic Peptide Recognized by Protective Antiviral CD8 T Cells
Authors: Holtappels Rafaela , Thomas Doris , Podlech Jürgen , Geginat Gernot , Steffens Hans-Peter , Reddehase Matthias J. 
Published: July 2002

ABSTRACTSeveral early genes of murine cytomegalovirus (MCMV) encode proteins that mediate immune evasion by interference with the major histocompatibility complex class I (MHC-I) pathway of antigen presentation to cytolytic T lymphocytes (CTL). Specifically, them152gene product gp37/40 causes retention of MHC-I molecules in the endoplasmic reticulum (ER)-Golgi intermediate compartment. Lack of MHC-I on the cell surface should activate natural killer (NK) cells recognizing the “missing self.” The retention, however, is counteracted by them04early gene product gp34, which binds to folded MHC-I molecules in the ER and directs the complex to the cell surface. It was thus speculated that gp34 might serve to silence NK cells and thereby complete the immune evasion of MCMV. In light of these current views, we provide here results demonstrating an in vivo role for gp34 in protective antiviral immunity. We have identified an antigenic nonapeptide derived from gp34 and presented by the MHC-I molecule Dd. Besides the immunodominant immediate-early nonapeptide consisting of IE1 amino acids 168-176 (IE1168-176), the early nonapeptide m04243-251is the second antigenic peptide described for MCMV. The primary immune response to MCMV generates significant m04-specific CD8 T-cell memory. Upon adoptive transfer into immunodeficient recipients, an m04-specific CTL line controls MCMV infection with an efficacy comparable to that of an IE1-specific CTL line. Thus, gp34 is the first noted early protein of MCMV that escapes viral immune evasion mechanisms. These data document that MCMV is held in check by a redundance of protective CD8 T cells recognizing antigenic peptides in different phases of viral gene expression.
Split-Intron Retroviral Vectors: Enhanced Expression with Improved Safety
Authors: Ismail Said I. , Kingsman Susan M. , Kingsman Alan J. , Uden Mark 
Published: July 2002

ABSTRACT
The inclusion of retrovirus-derived introns within retrovirus-based expression vectors leads to a fraction of the resulting transcripts being spliced. Such splicing has been shown to markedly improve expression (W. J. Krall et al., Gene Ther. 3:37–48, 1996). One way to improve upon this still further might involve the use of more efficient introns instead of those from the provirus. Currently, however, incorporation of such introns remains self-defeating since they are removed in the nucleus of the producer cell. In the past, elaborate ways to overcome this problem have included the use of alphaviruses to make the vector transcripts within the cytoplasm, thus avoiding the nuclear splicing machinery during vector production (K. J. Li and H. Garoff, Proc. Natl. Acad. Sci. USA 95:3650–3654, 1998). We now present a novel design for the inclusion of introns within a retroviral vector. In essence, this is achieved by exploiting the retroviral replication process to copy not only the U3 promoter but also a synthetic splice donor to the 5′-long-terminal-repeat position during reverse transcription. Once copied, synthesized transcripts then contain a splice donor at their 5′ end capable of interacting with a consensus splice acceptor engineered downstream of the packaging signal. Upon transduction, we demonstrate these vectors to produce enhanced expression from near fully spliced (and thus packaging signal minus) transcripts. The unique design of these high titer and high-expression retroviral vectors may be of use in a number of gene therapy applications.