bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induction of Protective Immune Responses against R5 Human Immunodeficiency Virus Type 1 (HIV-1) Infection in hu-PBL-SCID Mice by Intrasplenic Immunization with HIV-1-Pulsed Dendritic Cells: Possible Involvement of a Novel Factor of Human CD4+T-Cell Origin
Authors: Yoshida Atsushi , Tanaka Reiko , Murakami Tsutomu , Takahashi Yoshiaki , Koyanagi Yoshio , Nakamura Masataka , Ito Mamoru , Yamamoto Naoki , Tanaka Yuetsu 
Published: July 2003

ABSTRACTThe potential of a dendritic cell (DC)-based vaccine against human immunodeficiency virus type 1 (HIV-1) infection in humans was explored with SCID mice reconstituted with human peripheral blood mononuclear cells (PBMC). HIV-1-negative normal human PBMC were transplanted directly into the spleens of SCID mice (hu-PBL-SCID-spl mice) together with autologous mature DCs pulsed with either inactivated HIV-1 (strain R5 or X4) or ovalbumin (OVA), followed by a booster injection 5 days later with autologous DCs pulsed with the same respective antigens. Five days later, these mice were challenged intraperitoneally with R5 HIV-1JR-CSF. Analysis of infection at 7 days postinfection showed that the DC-HIV-1-immunized hu-PBL-SCID-spl mice, irrespective of the HIV-1 isolate used for immunization, were protected against HIV-1 infection. In contrast, none of the DC-OVA-immunized mice were protected. Sera from the DC-HIV-1- but not the DC-OVA-immunized mice inhibited the in vitro infection of activated PBMC and macrophages with R5, but not X4, HIV-1. Upon restimulation with HIV-1 in vitro, the human CD4+T cells derived from the DC-HIV-1-immunized mice produced a similar R5 HIV-1 suppressor factor. Neutralizing antibodies against human RANTES, MIP-1α, MIP-1β, alpha interferon (IFN-α), IFN-β, IFN-γ, interleukin-4 (IL-4), IL-10, IL-13, IL-16, MCP-1, MCP-3, tumor necrosis factor alpha (TNF-α), or TNF-β failed to reverse the HIV-1-suppressive activity. These results show that inactivated HIV-1-pulsed autologous DCs can stimulate splenic resident human CD4+T cells in hu-PBL-SCID-spl mice to produce a yet-to-be-defined, novel soluble factor(s) with protective properties against R5 HIV-1 infection.
Plasmid Chemokines and Colony-Stimulating Factors Enhance the Immunogenicity of DNA Priming-Viral Vector Boosting Human Immunodeficiency Virus Type 1 Vaccines
Authors: Barouch Dan H. , McKay Paul F. , Sumida Shawn M. , Santra Sampa , Jackson Shawn S. , Gorgone Darci A. , Lifton Michelle A. , Chakrabarti Bimal K. , Xu Ling , Nabel Gary J. , Letvin Norman L. 
Published: July 2003

ABSTRACTHeterologous “prime-boost” regimens that involve priming with plasmid DNA vaccines and boosting with recombinant viral vectors have been shown to elicit potent virus-specific cytotoxic T-lymphocyte responses. Increasing evidence, however, suggests that the utility of recombinant viral vectors in human populations will be significantly limited by preexisting antivector immunity. Here we demonstrate that the coadministration of plasmid chemokines and colony-stimulating factors with plasmid DNA vaccines markedly increases the immunogenicity of DNA prime-recombinant adenovirus serotype 5 (rAd5) boost and DNA prime-recombinant vaccinia virus (rVac) boost vaccine regimens in BALB/c mice. In mice with preexisting anti-Ad5 immunity, priming with the DNA vaccine alone followed by rAd5 boosting elicited only marginal immune responses. In contrast, cytokine-augmented DNA vaccine priming followed by rAd5 vector boosting was able to generate potent immune responses in mice with preexisting anti-Ad5 immunity. These data demonstrate that plasmid cytokines can markedly improve the immunogenicity of DNA prime-viral vector boost vaccine strategies and can partially compensate for antivector immunity.
Identification of the E9^E2C cDNA and Functional Characterization of the Gene Product Reveal a New Repressor of Transcription and Replication in Cottontail Rabbit Papillomavirus
Authors: Jeckel Sonja , Loetzsch Ekaterina , Huber Evamaria , Stubenrauch Frank , Iftner Thomas 
Published: July 2003

ABSTRACT
Cottontail rabbit papillomavirus (CRPV) genomes mutated in the trans-activation domain of the E2 protein, which stimulates both viral DNA replication and transcription, are severely impaired in their ability to induce tumors in New Zealand White rabbits. A number of papillomaviruses encode, in addition to full-length E2, a shortened E2 protein or an E2 protein fused to a short stretch of amino acids derived from the small E8 open reading frame that counteract the activities of E2. We identified and cloned the novel cDNA E9^E2C of CRPV from papillomas of New Zealand White and cottontail rabbits and characterized the functions of the encoded gene product. E9^E2C was shown to be a bona fide repressor of minimal viral promoters, with the E9 domain being essential for this activity, and to repress E1/E2-dependent replication of a CRPV origin construct. In addition, E9^E2C counteracted the transactivation effect of the full-length E2 on minimal promoters containing several E2 binding sites. To investigate the role of E9^E2C in tumorigenesis, we constructed two CRPV genomes mutated in E9^E2C. One, designated CRPV-E9atgmut-pLAII, contained a mutation in the unique start codon in the E9 open reading frame, and the second E9^E2C mutant was constructed by the introduction of a stop codon close to the splice donor site at nucleotide 3714 that additionally prevented the correct splicing of the transcript. When we infected New Zealand White rabbits with these constructs, we surprisingly noted no differences in tumor induction efficiency, viral genome copy number, and viral transcription in comparison to wild-type CRPV.
Stable High-Producer Cell Clone Expressing Virus-Like Particles of the Japanese Encephalitis Virus E Protein for a Second-Generation Subunit Vaccine
Authors: Kojima Asato , Yasuda Atsushi , Asanuma Hideki , Ishikawa Toyokazu , Takamizawa Akihisa , Yasui Kotaro , Kurata Takeshi 
Published: July 2003

ABSTRACTWe produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.5 μg per 104cells) to the internationally licensed JE vaccine JE-VAX. E-VLP production was stable after multiple cell passages and persisted over 1 year with 100% expressing cells without detectable cell fusion, apoptosis, or cell death, but was suspended when the cells grew to 100% confluency and contact inhibition occurred. Mice immunized with the purified J12#26 E-antigen without adjuvant developed high titers of neutralizing antibodies for at least 7 months and 100% protection against intraperitoneal challenge with 5 × 106PFU of JEV when examined according to the JE vaccine standardization protocol. These results suggest that the recombinant E-VLP antigen produced by the J12#26 cell clone is an effective, safe, and low-cost second-generation subunit JE vaccine.
Probing the Importance of tRNA Anticodon: Human Immunodeficiency Virus Type 1 (HIV-1) RNA Genome Complementarity with an HIV-1 That Selects tRNA
Glu
for Replication
Authors: Dupuy Lesley C. , Kelly Nathan J. , Elgavish Tricia E. , Harvey Stephen C. , Morrow Casey D. 
Published: July 2003

ABSTRACT

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at the primer binding site (PBS) that is complementary to the 3′-terminal nucleotides of tRNA
3
Lys
. Why all known strains of HIV-1 select tRNA
3
Lys
for replication is unknown. Previous studies on the effect of altering the PBS of HIV-1 on replication identified an HIV-1 with a PBS complementary to tRNA
Glu
. Since the virus was not initially designed to use tRNA
Glu
, the virus had selected tRNA
Glu
from the intracellular pool of tRNA for use in replication. Further characterization of HIV-1 that uses tRNA
Glu
may provide new insights into the preference for tRNA
3
Lys
. HIV-1 constructed with the PBS complementary to tRNA
Glu
was more stable than HIV-1 with the PBS complementary to tRNA
Met
or tRNA
His
; however, all of these viruses eventually reverted back to using tRNA
3
Lys
following growth in SupT1 cells or peripheral blood mononuclear cells (PBMCs). New HIV-1 mutants with nucleotides in U5 complementary to the anticodon of tRNA
Glu
remained stable when grown in SupT1 cells or PBMCs, although the mutants grew more slowly than the wild-type virus. Sequence analysis of the U5 region and the PBS revealed additional mutations predicted to further promote tRNA-viral genome interaction. The results support the importance of the tRNA anticodon-genome interaction in the selection of the tRNA primer and highlight the fact that unique features of tRNA
3
Lys
are exploited by HIV-1 for selection as the reverse transcription primer.
A Hepadnavirus Regulatory Element Enhances Expression of a Type 2 Bovine Viral Diarrhea Virus E2 Protein from a Bovine Herpesvirus 1 Vector
Authors: Wang Lingshu , Menon Sreekumar , Bolin Steven R. , Bello Leonard J. 
Published: July 2003

ABSTRACTRecently, the possibility of using virus vectors to immunize cattle against selected bovine viral diarrhea virus (BVDV) genes has gained widespread interest. However, when we attempted to express the E2 protein from type 2 (890 strain) BVDV in a bovine herpesvirus 1 (BHV1) vector, we observed that expression was poor. This often happens when genes from a cytoplasmic virus are expressed in the cell nucleus. To counter this effect, we attempted to enhance expression by a strategy employed by viruses. RNAs of retroviruses and hepadnaviruses containcis-acting elements that facilitate expression of RNAs that otherwise are degraded or retained within the nucleus. In Mason-Pfizer monkey virus, the required RNA sequence element is known as a constitutive transport element (CTE). A related element from woodchuck hepatitis virus is known as the woodchuck posttranscriptional regulatory element (WPRE). We tested the ability of the CTE, the WPRE, and introns to enhance expression of E2. All three elements stimulated expression of E2 from plasmids. The combination of the WPRE and an intron yielded the highest level of E2 expression in plasmids. However, when E2 was expressed from a BHV1 vector, the presence of an intron was inhibitory. In contrast, the WPRE was very efficient at stimulating E2 expression from a BHV1 vector. This result represents the first expression of a type 2 BVDV E2 protein from a mammalian virus vector and raises the possibility that the WPRE may provide a general method of enhancing foreign gene expression from BHV1 and other herpesvirus vectors.
Simian Immunodeficiency Virus Infection in Neonatal Macaques
Authors: Veazey Ronald S. , Lifson Jeffrey D. , Pandrea Ivona , Purcell Jeannette , Piatak Michael , Lackner Andrew A. 
Published: July 2003

ABSTRACTChildren with human immunodeficiency virus infection often have higher viral loads and progress to AIDS more rapidly than adults. Since the intestinal tract is a major site of early viral replication and CD4+T-cell depletion in adults, we examined the effects of simian immunodeficiency virus (SIV) on both peripheral and intestinal lymphocytes from 13 neonatal macaques infected with SIVmac239. Normal neonates had more CD4+T cells and fewer CD8+T cells in all tissues than adults. Surprisingly, neonates had substantial percentages of CD4+T cells with an activated, memory phenotype (effector CD4+T cells) in the lamina propria of the intestine compared to peripheral lymphoid tissues, even when examined on the day of birth. Moreover, profound and selective depletion of jejunum lamina propria CD4+T cells occurred in neonatal macaques within 21 days of infection, which was preceded by large numbers of SIV-infected cells in this compartment. Furthermore, neonates with less CD4+T-cell depletion in tissues tended to have higher viral loads. The persistence of intestinal lamina propria CD4+T cells in some neonates with high viral loads suggests that increased turnover and/or resistance to CD4+T-cell loss may contribute to the higher viral loads and increased severity of disease in neonatal hosts.
Adaptation of Puumala Hantavirus to Cell Culture Is Associated with Point Mutations in the Coding Region of the L Segment and in the Noncoding Regions of the S Segment
Authors: Nemirov Kirill , Lundkvist Åke , Vaheri Antti , Plyusnin Alexander 
Published: July 2003

ABSTRACT
We previously developed a model for studies on hantavirus host adaptation and initiated genetic analysis of Puumala virus variants passaged in colonized bank voles and in cultured Vero E6 cells. With the data presented in this paper, the sequence comparison of the wild-type and Vero E6-adapted variants of Puumala virus, strain Kazan, has been completed. The only amino acid substitution that distinguished the two virus variants was found in the L protein, Ser versus Phe at position 2053. Another mutation found in the L segment, the silent transition C1053U, could result from the selection of a variant with altered L RNA folding. Nucleotide substitutions observed in individual L cDNA clones, most of them A→G and U→C transitions, suggested that the population of L RNA molecules is represented by quasispecies. The mutation frequency in the L segment quasispecies appeared to be similar to the corresponding values for the S and M quasispecies. Analysis of the cDNA clones with the complete S segment sequences from passage 20 confirmed our earlier conclusion that the cell-adapted genotype of the virus is represented mostly by variants with mutated S segment noncoding regions. However, the spectrum of the S segment quasispecies appeared to be changing, suggesting that, after the initial adaptation (passages 1 to 11), the viral population is still being driven by selection for variants with higher fitness.
The Coronavirus Spike Protein Is a Class I Virus Fusion Protein: Structural and Functional Characterization of the Fusion Core Complex
Authors: Bosch Berend Jan , van der Zee Ruurd , de Haan Cornelis A. M. , Rottier Peter J. M. 
Published: July 2003

ABSTRACT

Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in
Escherichia coli
. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure ∼14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein.
Strain Variation in an Emerging Iridovirus of Warm-Water Fishes
Authors: Goldberg Tony L. , Coleman David A. , Grant Emily C. , Inendino Kate R. , Philipp David P. 
Published: July 2003

ABSTRACT
Although iridoviruses vary widely within and among genera with respect to their host range and virulence, variation within iridovirus species has been less extensively characterized. This study explores the nature and extent of intraspecific variation within an emerging iridovirus of North American warm-water fishes, largemouth bass virus (LMBV). Three LMBV isolates recovered from three distinct sources differed genetically and phenotypically. Genetically, the isolates differed in the banding patterns generated from amplified fragment length polymorphism analysis but not in their DNA sequences at two loci of different degrees of evolutionary stability. In vitro, the isolates replicated at identical rates in cell culture, as determined by real-time quantitative PCR of viral particles released into suspension. In vivo, the isolates varied over fivefold in virulence, as measured by the rate at which they induced mortality in juvenile largemouth bass. This variation was reflected in the viral loads of exposed fish, measured using real-time quantitative PCR; the most virulent viral strain also replicated to the highest level in fish. Together, these results justify the designation of these isolates as different strains of LMBV. Strain variation in iridoviruses could help explain why animal populations naturally infected with iridovirus pathogens vary so extensively in their clinical responses to infection. The results of this study are especially relevant to emerging iridoviruses of aquaculture systems and wildlife.
Feline Immunodeficiency Virus Orf-A Is Required for Virus Particle Formation and Virus Infectivity
Authors: Gemeniano Malou C. , Sawai Earl T. , Leutenegger Christian M. , Sparger Ellen E. 
Published: July 2003

ABSTRACT

The
orf-A
(
orf-2
) gene of feline immunodeficiency virus (FIV) is a small open reading frame predicted to encode a 77-amino-acid protein that contains putative domains similar to those of the ungulate lentiviral Tat protein. Orf-A is reported to be critical for efficient viral replication in vitro and in vivo. A series of FIV-pPPR-derived proviruses with in-frame deletions and point mutations within
orf-A
were constructed and tested for replication in feline lymphoid cells. Orf-A mutant proviruses were also tested for viral gene and protein expression, viral particle formation, and virion infectivity. Deletions within
orf-A
severely restricted FIV replication in feline peripheral blood mononuclear cells (PBMC) and interleukin-2-dependent T-cell lines. In addition, substitutions of alanines for leucines in the putative leucine-rich domain, for cysteines in the putative cysteine-rich domain, and for a tryptophan at position 43 in Orf-A restricted the replication of FIV mutants. Deletions and point mutations in
orf-A
imposed a small effect or no effect on FIV long-terminal-repeat-driven viral gene expression and had no effect on viral protein expression. However, release of cell-free, virion-associated viral RNA in supernatants from cells transfected with
orf-A
mutant proviruses was severely restricted but was rescued by cotransfection with a wild-type Orf-A expression vector. In addition, virions derived from
orf-A
mutant proviruses expressed reduced infectivity for feline PBMC. Our findings suggest that Orf-A functions involve multiple steps of the FIV life cycle including both virion formation and infectivity. Furthermore, these observations suggest that Orf-A represents an FIV-encoded analog more similar to the accessory gene
vpr
,
vpu
, or
nef
than to the regulatory gene
tat
encoded by the primate lentiviruses.
De Novo Synthesis of Negative-Strand RNA by Dengue Virus RNA-Dependent RNA Polymerase In Vitro: Nucleotide, Primer, and Template Parameters
Authors: Nomaguchi Masako , Ackermann Matt , Yon Changsuek , You Shihyun , Padmanbhan R. 
Published: July 2003

ABSTRACT
By using a purified dengue virus RNA-dependent RNA polymerase and a subgenomic 770-nucleotide RNA template, it was shown previously that the ratio of the de novo synthesis product to hairpin product formed was inversely proportional to increments of assay temperatures (20 to 40°C). In this study, the components of the de novo preinitiation complex are defined as ATP, a high concentration of GTP (500 μM), the polymerase, and the template RNA. Even when the 3′-terminal sequence of template RNA was mutated from -GGUUCU-3′ to -GGUUUU-3′, a high GTP concentration was required for de novo initiation, suggesting that high GTP concentration plays a conformational role. Furthermore, utilization of synthetic primers by the polymerase indicated that AGAA is the optimal primer whereas AG, AGA, and AGAACC were inefficient primers. Moreover, mutational analysis of the highly conserved 3′-terminal dinucleotide CU of the template RNA indicated that change of the 3′-terminal nucleotide from U to C reduced the efficiency about fivefold. The order of preference for the 3′-terminal nucleotide, from highest to lowest, is U, A∼G, and C. However, change of the penultimate nucleotide from C to U did not affect the template activity. A model consistent with these results is that the active site of the polymerase switches from a “closed” form, catalyzing de novo initiation through synthesis of short primers, to an “open” form for elongation of a double-stranded template-primer.
Development of Recombinant Vesicular Stomatitis Viruses That Exploit Defects in Host Defense To Augment Specific Oncolytic Activity
Authors: Obuchi Masatsugu , Fernandez Marilyn , Barber Glen N. 
Published: July 2003

ABSTRACTVesicular stomatitis virus (VSV) is a negative-stranded RNA virus normally sensitive to the antiviral actions of alpha/beta interferon (IFN-α/β). Recently, we reported that VSV replicates to high levels in many transformed cells due, in part, to susceptible cells harboring defects in the IFN system. These observations were exploited to demonstrate that VSV can be used as a viral oncolytic agent to eradicate malignant cells in vivo while leaving normal tissue relatively unaffected. To attempt to improve the specificity and efficacy of this system as a potential tool in gene therapy and against malignant disease, we have genetically engineered VSV that expresses the murine IFN-β gene. The resultant virus (VSV-IFNβ) was successfully propagated in cells not receptive to murine IFN-α/β and expressed high levels of functional heterologous IFN-β. In normal murine embryonic fibroblasts (MEFs), the growth of VSV-IFNβ was greatly reduced and diminished cytopathic effect was observed due to the production of recombinant IFN-β, which by functioning in a manner involving autocrine and paracrine mechanisms induced an antiviral effect, preventing virus growth. However, VSV-IFNβ grew to high levels and induced the rapid apoptosis of transformed cells due to defective IFN pathways being prevalent and thus unable to initiate proficient IFN-mediated host defense. Importantly, VSV expressing the human IFN-β gene (VSV-hIFNβ) behaved comparably and, while nonlytic to normal human cells, readily killed their malignant counterparts. Similar to our in vitro observations, following intravenous and intranasal inoculation in mice, recombinant VSV (rVSV)-IFNβ was also significantly attenuated compared to wild-type VSV or rVSV expressing green fluorescent protein. However, VSV-IFNβ retained propitious oncolytic activity against metastatic lung disease in immunocompetent animals and was able to generate robust antitumor T-cell responses. Our data indicate that rVSV designed to exploit defects in mechanisms of host defense can provide the basis for new generations of effective, specific, and safer viral vectors for the treatment of malignant and other disease.