bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoreactivity of Intact Virions of Human Immunodeficiency Virus Type 1 (HIV-1) Reveals the Existence of Fewer HIV-1 Immunotypes than Genotypes
Authors: Nyambi Phillipe N. , Nádas Arthur , Mbah Henry A. , Burda Sherri , Williams Constance , Gorny Miroslaw K. , Zolla-Pazner Susan
Published: July 2002

In order to protect against organisms that exhibit significant genetic variation, polyvalent vaccines are needed. Given the extreme variability of human immunodeficiency virus type 1 (HIV-1), it is probable that a polyvalent vaccine will also be needed for protection from this virus. However, to understand how to construct a polyvalent vaccine, serotypes or immunotypes of HIV must be identified. In the present study, we have examined the immunologic relatedness of intact, native HIV-1 primary isolates of group M, clades A to H, with human monoclonal antibodies (MAbs) directed at epitopes in the V3, C5, and gp41 cluster I regions of the envelope glycoproteins, since these regions are well exposed on the virion surface. Multivariate analysis of the binding data revealed three immunotypes of HIV-1 and five MAb groups useful for immunotyping of the viruses. The analysis revealed that there are fewer immunotypes than genotypes of HIV and that clustering of the isolates did not correlate with either genotypes, coreceptor usage (CCR5 and CXCR4), or geographic origin of the isolates. Further analysis revealed distinct MAb groups that bound preferentially to HIV-1 isolates belonging to particular immunotypes or that bound to all three immunotypes; this demonstrates that viral immunotypes identified by mathematical analysis are indeed defined by their immunologic characteristics. In summary, these results indicate (i) that HIV-1 immunotypes can be defined, (ii) that constellations of epitopes that are conserved among isolates belonging to each individual HIV-1 immunotype exist and that these distinguish each of the immunotypes, and (iii) that there are also epitopes that are routinely shared by all immunotypes.
Episomal Segregation of the Adenovirus Enhancer Sequence by Conditional Genome Rearrangement Abrogates Late Viral Gene Expression
Authors: Wang Xinzhong , Zeng Weike , Murakawa Masahiro , Freeman Mason W. , Seed Brian
Published: July 2002

We have constructed a recombinant adenovirus gene delivery system that is capable of undergoing growth phase-dependent site-specific recombination. When propagated in 293 producer cells, the vector retains its linear double-stranded form and can be propagated to high titer and purified by conventional procedures. Upon introduction into target cells, the viral chromosome undergoes cyclization to generate an autonomously replicating circular episome and a detached linear fragment. The viral enhancer and reporter gene segregate with the circular episome, which contains no adenovirus open reading frames. The effect of rearrangement of adenovirus gene expression was assessed by quantitative reverse transcription-PCR measurement of the abundance of transcripts encoding the tripartite leader sequence (TPL) of the major late promoter. Whereas nonrearranging viruses produced approximately 10
4
TPL transcripts per 10
6
infecting genomes in the HepG2 liver cell line, no transcripts were detectable in the same cells infected with comparable levels of circularizing vector. Because no helper virus is required to propagate these vectors, the problems of recombination with and contamination by helper virus are eliminated. We also present an efficient and reliable method for generating recombinant adenoviruses.
Characterization of a Virtually Full-Length Human Immunodeficiency Virus Type 1 Genome of a Prevalent Intersubtype (C/B′) Recombinant Strain in China
Authors: Su Ling , Graf Marcus , Zhang Yuanzhi , von Briesen Hagen , Xing Hui , Köstler Josef , Melzl Holger , Wolf Hans , Shao Yiming , Wagner Ralf
Published: July 2002

A molecular epidemiology study was conducted among more than 100 human immunodeficiency virus type 1 (HIV-1) subtype C seropositive intravenous drug users (IDUs) from China. Genotyping based on the envelope C2V3 coding region revealed the highest homology of the most prevalent virus strains circulating throughout China to subtype C sequences of Indian origin. Based on these results, a virtually full-length genome representing the most prevalent class of clade C strains circulating throughout China was directly amplified from peripheral blood mononuclear cells of a selected HIV-infected IDU and subcloned. Sequence analysis identified a mosaic structure, suggesting extensive intersubtype recombination events between genomes of the prevalent clade C and (B′)-subtype Thai virus strains of that geographic region. Recombinant Identification Program analysis and phylogenetic bootstrapping suggested that there were 10 breakpoints (i) in the
gag-pol
coding region, (ii) in
vpr
and at the 3′ end of the
vpu
gene, and (iii) in the
nef
open reading frame. (B′)-sequences therefore include (i) several insertions in the
gag-pol
coding region; (ii) 3′-
vpr
, the complete
vpu
gene, and the first exons of
tat
and
rev
; and (iii) the 5′ half of the
nef
gene. Breakpoints located in the
vpr/vpu
coding region as well as in the
nef
gene of 97cn54 were found at almost identical positions of all subtype C strains isolated from IDUs living in different areas of China, suggesting a common ancestor for the C/B′ recombinant strains. More than 50% of well-defined subtype B-derived cytotoxic T-lymphocyte epitopes within Gag and Pol and 10% of the known epitopes in Env were found to exactly match sequences within in this clade C/B′ chimeric reference strain. These results may substantially facilitate a biological comparison of clade C-derived reference strains as well as the generation of useful reagents supporting vaccine-related efforts in China.
Effects of Tumor Necrosis Factor Alpha on Sin Nombre Virus Infection In Vitro
Authors: Khaiboullina Svetlana F. , Netski Dale M. , Krumpe Peter , St. Jeor Stephen C.
Published: July 2002

Previous data indicate that immune mechanisms may be involved in developing capillary leakage during Sin Nombre virus (SNV) infection. Therefore, we investigated production of tumor necrosis factor alpha (TNF-α) by human alveolar macrophages and human umbilical vein endothelial cells (HUVEC) after infection with SNV. In addition, we examined the effect of TNF-α on HUVEC monolayer leakage. Our results reveal that although TNF-α decreases accumulation of viral nucleoproteins, TNF-α levels do not change in SNV-infected cells. In addition, supernatants from SNV-infected human alveolar macrophages did not cause a significant increase in endothelial monolayer permeability.
Alpha/Beta Interferons Potentiate Virus-Induced Apoptosis through Activation of the FADD/Caspase-8 Death Signaling Pathway
Authors: Balachandran Siddharth , Roberts P. Christopher , Kipperman Todd , Bhalla Kapil N. , Compans Richard W. , Archer David R. , Barber Glen N.
Published: July 2002

Interferon (IFN) mediates its antiviral effects by inducing a number of responsive genes, including the double-stranded RNA (dsRNA)-dependent protein kinase, PKR. Here we report that inducible overexpression of functional PKR in murine fibroblasts sensitized cells to apoptosis induced by influenza virus, while in contrast, cells expressing a dominant-negative variant of PKR were completely resistant. We determined that the mechanism of influenza virus-induced apoptosis involved death signaling through FADD/caspase-8 activation, while other viruses such as vesicular stomatitis virus (VSV) and Sindbis virus (SNV) did not significantly provoke PKR-mediated apoptosis but did induce cytolysis of fibroblasts via activation of caspase-9. Significantly, treatment with IFN-α/β greatly sensitized the fibroblasts to FADD-dependent apoptosis in response to dsRNA treatment or influenza virus infection but completely protected the cells against VSV and SNV replication in the absence of any cellular destruction. The mechanism by which IFN increases the cells' susceptibility to lysis by dsRNA or certain virus infection is by priming cells to FADD-dependent apoptosis, possibly by regulating the activity of the death-induced signaling complex (DISC). Conversely, IFN is also able to prevent the replication of viruses such as VSV that avoid triggering FADD-mediated DISC activity, by noncytopathic mechanisms, thus preventing destruction of the cell.
Infectious Entry Pathway of Adeno-Associated Virus and Adeno-Associated Virus Vectors
Authors: Bartlett Jeffrey S. , Wilcher Rose , Samulski R. Jude
Published: July 2002

We have investigated the infectious entry pathway of adeno-associated virus (AAV) and recombinant AAV vectors by assessing AAV-mediated gene transfer and by covalently conjugating fluorophores to AAV and monitoring entry by fluorescence microscopy. We examined AAV entry in HeLa cells and in HeLa cell lines which inducibly expressed a dominant interfering mutant of dynamin. The data demonstrate that AAV internalizes rapidly by standard receptor-mediated endocytosis from clathrin-coated pits (half-time <10 min). The lysosomotropic agents ammonium chloride and bafilomycin A
1
prevent AAV-mediated gene transfer when present during the first 30 min after the onset of endocytosis, indicating that AAV escapes from early endosomes yet requires an acidic environment for penetration into the cytosol. Following release from the endosome, AAV rapidly moves to the cell nucleus and accumulates perinuclearly beginning within 30 min after the onset of endocytosis. We present data indicating that escape of AAV from the endosome and trafficking of viral particles to the nucleus are unaffected by the presence of adenovirus, the primary helper virus for a productive AAV infection. Within 2 h, viral particles could be detected within the cell nucleus, suggesting that AAV enters the nucleus prior to uncoating. Interestingly, the majority of the intracellular virus particles remain in a stable perinuclear compartment even though gene expression from nuclear AAV genomes can be detected. This suggests that the process of nuclear entry is rate limiting or that AAV entry involves multiple pathways. Nevertheless, these data establish specific points in the AAV infectious entry process and have allowed the generation of a model for future expansion to specific cell types and AAV vector analysis in vivo.
Identification of a Boundary Domain Adjacent to the Potent Human Cytomegalovirus Enhancer That Represses Transcription of the Divergent UL127 Promoter
Authors: Angulo Ana , Kerry David , Huang Huang , Borst Eva-Maria , Razinsky Alison , Wu Jun , Hobom Urs , Messerle Martin , Ghazal Peter
Published: July 2002

Transcriptional repression within a complex modular promoter may play a key role in determining the action of enhancer elements. In human cytomegalovirus, the major immediate-early promoter (MIEP) locus contains a highly potent and complex modular enhancer. Evidence is presented suggesting that sequences of the MIEP between nucleotide positions −556 and −673 function to prevent transcription activation by enhancer elements from the UL127 open reading frame divergent promoter. Transient transfection assays of reporter plasmids revealed repressor sequences located between nucleotides −556 and −638. The ability of these sequences to confer repression in the context of an infection was shown using recombinant viruses generated from a bacterial artificial chromosome containing an infectious human cytomegalovirus genome. In addition to repressor sequences between −556 and −638, infection experiments using recombinant virus mutants indicated that sequences between −638 and −673 also contribute to repression of the UL127 promoter. On the basis of in vitro transcription and transient transfection assays, we further show that interposed viral repressor sequences completely inhibit enhancer-mediated activation of not only the homologous but also heterologous promoters. These and other experiments suggest that repression involves an interaction of host-encoded regulatory factors with defined promoter sequences that have the property of proximally interfering with upstream enhancer elements in a chromatin-independent manner. Altogether, our findings establish the presence of a boundary domain that efficiently blocks enhancer-promoter interactions, thus explaining how the enhancer can work to selectively activate the MIEP.
Evidence for Recombination of Live, Attenuated Immunodeficiency Virus Vaccine with Challenge Virus to a More Virulent Strain
Authors: Gundlach Björn R. , Lewis Mark G. , Sopper Sieghart , Schnell Tanja , Sodroski Joseph , Stahl-Hennig Christiane , Überla Klaus
Published: July 2002

Live, attenuated immunodeficiency virus vaccines, such as
nef
deletion mutants, are the most effective vaccines tested in the simian immunodeficiency virus (SIV) macaque model. In two independent studies designed to determine the breadth of protection induced by live, attenuated SIV vaccines, we noticed that three of the vaccinated macaques developed higher set point viral load levels than unvaccinated control monkeys. Two of these vaccinated monkeys developed AIDS, while the control monkeys infected in parallel remained asymptomatic. Concomitant with an increase in viral load, a recombinant of the vaccine virus and the challenge virus could be detected. Therefore, the emergence of more-virulent recombinants of live, attenuated immunodeficiency viruses and less-aggressive wild-type viruses seems to be an additional risk of live, attenuated immunodeficiency virus vaccines.
Identification of a Conserved Residue of Foamy Virus Gag Required for Intracellular Capsid Assembly
Authors: Eastman Scott W. , Linial Maxine L.
Published: July 2002

In contrast to all retroviruses but similar to the hepatitis B virus, foamy viruses (FV) require expression of the envelope protein for budding of intracellular capsids from the cell, suggesting a specific interaction between the Gag and Env proteins. Capsid assembly occurs in the cytoplasm of infected cells in a manner similar to that for the B- and D-type viruses; however, in contrast to these retroviruses, FV Gag lacks an N-terminal myristylation signal and capsids are not targeted to the plasma membrane (PM). We have found that mutation of an absolutely conserved arginine (Arg) residue at position 50 to alanine (R50A) of the simian foamy virus SFV cpz(hu) inhibits proper capsid assembly and abolishes viral budding even in the presence of the envelope (Env) glycoproteins. Particle assembly and extracellular release of virus can be restored to this mutant with the addition of an N-terminal Src myristylation signal (Myr-R50A), presumably by providing an alternate site for assembly to occur at the PM. In addition, the strict requirement of Env expression for capsid budding can be bypassed by addition of a PM-targeting signal to Gag. These results suggest that intracellular capsid assembly may be mediated by a signal akin to the cytoplasmic targeting and retention signal CTRS found in Mason-Pfizer monkey virus and that FV Gag has the inherent ability to assemble capsids at multiple sites like conventional retroviruses. The necessity of Env expression for particle egress is most probably due to the lack of a membrane-targeting signal within FV Gag to direct capsids to the PM for release and indicates that Gag-Env interactions are essential to drive particle budding.
Influenza B and C Virus NEP (NS2) Proteins Possess Nuclear Export Activities
Authors: Paragas Jason , Talon Julie , O'Neill Robert E. , Anderson D. Karl , Garcı́a-Sastre Adolfo , Palese Peter
Published: July 2002

Nucleocytoplasmic transport of viral ribonucleoproteins (vRNPs) is an essential aspect of the replication cycle for influenza A, B, and C viruses. These viruses replicate and transcribe their genomes in the nuclei of infected cells. During the late stages of infection, vRNPs must be exported from the nucleus to the cytoplasm prior to transport to viral assembly sites on the cellular plasma membrane. Previously, we demonstrated that the influenza A virus nuclear export protein (NEP, formerly referred to as the NS2 protein) mediates the export of vRNPs. In this report, we suggest that for influenza B and C viruses the nuclear export function is also performed by the orthologous NEP proteins (formerly referred to as the NS2 protein). The influenza virus B and C NEP proteins interact in the yeast two-hybrid assay with a subset of nucleoporins and with the Crm1 nuclear export factor and can functionally replace the effector domain from the human immunodeficiency virus type 1 Rev protein. We established a plasmid transfection system for the generation of virus-like particles (VLPs) in which a functional viral RNA-like chloramphenicol acetyltransferase (CAT) gene is delivered to a new cell. VLPs generated in the absence of the influenza B virus NEP protein were unable to transfer the viral RNA-like CAT gene to a new cell. From these data, we suggest that the nuclear export of the influenza B and C vRNPs are mediated through interaction between NEP proteins and the cellular nucleocytoplasmic export machinery.
Plasminogen-Binding Activity of Neuraminidase Determines the Pathogenicity of Influenza A Virus
Authors: Goto Hideo , Wells Krisna , Takada Ayato , Kawaoka Yoshihiro
Published: July 2002

When expressed in vitro, the neuraminidase (NA) of A/WSN/33 (WSN) virus binds and sequesters plasminogen on the cell surface, leading to enhanced cleavage of the viral hemagglutinin. To obtain direct evidence that the plasminogen-binding activity of the NA enhances the pathogenicity of WSN virus, we generated mutant viruses whose NAs lacked plasminogen-binding activity because of a mutation at the C terminus, from Lys to Arg or Leu. In the presence of trypsin, these mutant viruses replicated similarly to wild-type virus in cell culture. By contrast, in the presence of plasminogen, the mutant viruses failed to undergo multiple cycles of replication while the wild-type virus grew normally. The mutant viruses showed attenuated growth in mice and failed to grow at all in the brain. Furthermore, another mutant WSN virus, possessing an NA with a glycosylation site at position 130 (146 in N2 numbering), leading to the loss of neurovirulence, failed to grow in cell culture in the presence of plasminogen. We conclude that the plasminogen-binding activity of the WSN NA determines its pathogenicity in mice.
Murine Cytomegalovirus CC Chemokine Homolog MCK-2 (m131-129) Is a Determinant of Dissemination That Increases Inflammation at Initial Sites of Infection
Authors: Saederup Noah , Aguirre Shirley A. , Sparer Timothy E. , Bouley Donna M. , Mocarski Edward S.
Published: July 2002

The murine cytomegalovirus CC chemokine homolog MCK-2 (m131-129) is an important determinant of dissemination during primary infection. Reduced peak levels of viremia at day 5 were followed by reduced levels of virus in salivary glands starting at day 7 whenmckinsertion (RM461) and point (RM4511) mutants were compared tomck-expressing viruses. A dramatic MCK-2-enhanced inflammation occurred at the inoculation site over the first few days of infection, preceding viremia. The data further reinforce the role of MCK-2 as a proinflammatory signal that recruits leukocytes to increase the efficiency of viral dissemination in the host.
The 5′ Untranslated Region of
Rhopalosiphum padi
Virus Contains an Internal Ribosome Entry Site Which Functions Efficiently in Mammalian, Plant, and Insect Translation Systems
Authors: Woolaway Kathryn E. , Lazaridis Konstantinos , Belsham Graham J. , Carter Michael J. , Roberts Lisa O.
Published: July 2002

Rhopalosiphum padi
virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of
Picornaviridae
, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5′ UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5′ UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, and
Drosophila
in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5′ IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.
Comparison of Polymerase Subunits from Double-Stranded RNA Bacteriophages
Authors: Yang Hongyan , Makeyev Eugene V. , Bamford Dennis H.
Published: July 2002

The family
Cystoviridae
comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the
Cystoviridae
members, bacteriophage φ6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3′ termini from the φ6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from φ6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the φ6-related bacteriophages φ8 and φ13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from φ6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3′-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription in
Cystoviridae
is controlled by the template specificity of the polymerase subunit.
Restoration of Function of Carboxy-Terminally Truncated Pseudorabies Virus Glycoprotein B by Point Mutations in the Ectodomain
Authors: Nixdorf Ralf , Klupp Barbara G. , Mettenleiter Thomas C.
Published: July 2002

Glycoprotein B (gB) of pseudorabies virus (PrV) is essential for virus entry into target cells and direct viral cell-to-cell spread. Recently, we described a carboxy-terminally truncated derivative of PrV gB, gB-007, which was inefficiently incorporated into virions, was unable to complement infectivity, but was fully capable of restoring direct viral cell-to-cell spread of gB-negative PrV (R. Nixdorf, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 74:7137–7145, 2000). Since recombinant PrV-007, which expresses gB-007 instead of wild-type gB, was able to spread directly from cell to cell, we attempted to obtain compensatory mutations leading to restoration of the entry defect by performing serial passages in cell culture. This procedure has previously been used to successfully restore entry defects in gD- or gL-deficient PrV mutants. From an initial titer of 100 PFU per ml in the supernatant, titers increased, reaching wild-type levels of up to 10
7
PFU after ca. 20 passages. One single-plaque isolate of the passaged mutant, designated PrV-007Pass, was further characterized. PrV-007Pass gB was efficiently incorporated into the viral envelope and restored infectivity to a gB-negative PrV mutant, PrV-gB
−
. Interestingly, localization of PrV-007Pass gB in the plasma membrane was similar to that of PrV-007. In contrast, wild-type gB is mainly found in intracellular vesicles. Marker rescue experiments and
trans
-complementation assays demonstrated the presence of compensatory mutations within the gB gene of PrV-007Pass. DNA sequencing revealed two point mutations in the gB open reading frame of PrV-007Pass, resulting in amino acid substitutions at positions 305 and 744 of gB, both of which are required for compensation of the defect in PrV-007. Our data again demonstrate the power of reversion analysis of herpesviruses and suggest that cytosolic and ectodomains play a role in incorporation of gB into virions.
Recovery of Infectious Pariacoto Virus from cDNA Clones and Identification of Susceptible Cell Lines
Authors: Johnson Karyn N. , Ball L. Andrew
Published: July 2002

Pariacoto virus (PaV) is a nodavirus that was recently isolated in Peru from the Southern armyworm,
Spodoptera eridania
. Virus particles are non enveloped and about 30 nm in diameter and have
T
=3 icosahedral symmetry. The 3.0-Å crystal structure shows that about 35% of the genomic RNA is icosahedrally ordered, with the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that underlie the inner surface of the capsid. The PaV genome comprises two single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor α. In order to apply molecular genetics to the structure and assembly of PaV, we identified susceptible cell lines and developed a reverse genetic system for this virus. Cell lines that were susceptible to infection by PaV included those from
Spodoptera exigua, Helicoverpa zea
and
Aedes albopictus
, whereas cells from
Drosophila melanogaster
and
Spodoptera frugiperda
were refractory to infection. To recover virus from molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were cotranscribed by T7 RNA polymerase in baby hamster kidney cells that expressed T7 RNA polymerase. Lysates of these cells were infectious both for cultured cells from
Helicoverpa zea
(corn earworm) and for larvae of
Galleria mellonella
(greater wax moth). The combination of infectious cDNA clones, cell culture infectivity, and the ability to produce milligram amounts of virus allows the application of DNA-based genetic methods to the study of PaV structure and assembly.
Two Functionally Distinct Forms of a Retroviral Receptor Explain the Nonreciprocal Receptor Interference among Subgroups B, D, and E Avian Leukosis Viruses
Authors: Adkins Heather B. , Blacklow Stephen C. , Young John A. T.
Published: July 2002

Subgroups B, D, and E avian leukosis viruses (ALV-B, -D, and -E) share the same chicken receptor, TVBS1, a tumor necrosis factor receptor (TNFR)-related protein. These viruses, however, exhibit nonreciprocal receptor interference (NRI): cells preinfected with ALV-B or ALV-D are resistant to superinfection by viruses of all three subgroups, whereas those pre-infected by ALV-E are resistant only to superinfection by other subgroup E viruses. In this study, we investigated the basis of this phenomenon by characterizing the interaction of TVBS1with ALV-B Env or ALV-E Env. Sequential immunoprecipitation analysis using surface envelope immunoglobulin fusion proteins revealed the existence of two separate types of TVBS1that are encoded by the same cDNA clone. One form, designated the type 1 receptor, is specific for ALV-B and ALV-E. The other form, the type 2 receptor, is specific for ALV-B. We show that a protein consisting of only the first and second extracellular cysteine-rich domains of TVBS1is capable of forming both receptor types. However, the third extracellular cysteine-rich domain is required for efficient formation of the type 1 receptor. We also demonstrate that heterogeneous N-linked glycosylation cannot explain the difference in activities of the two receptor types. The existence of two types of TVBS1explains the NRI pattern between ALV-B and -E: subgroup B viruses establish receptor interference with both receptor types, whereas subgroup E viruses interfere only with the type 1 receptor, leaving the type 2 receptor available to mediate subsequent rounds of ALV-B entry. The formation of a TVB receptor type that is specific for cytopathic ALV may also have important implications for understanding how some subgroups of ALV cause cell death.
Genetic Targeting of an Adenovirus Vector via Replacement of the Fiber Protein with the Phage T4 Fibritin
Authors: Krasnykh Victor , Belousova Natalya , Korokhov Nikolay , Mikheeva Galina , Curiel David T.
Published: July 2002

The utility of adenovirus (Ad) vectors for gene therapy is restricted by their inability to selectively transduce disease-affected tissues. This limitation may be overcome by the derivation of vectors capable of interacting with receptors specifically expressed in the target tissue. Previous attempts to alter Ad tropism by genetic modification of the Ad fiber have had limited success due to structural conflicts between the fiber and the targeting ligand. Here we present a strategy to derive an Ad vector with enhanced targeting potential by a radical replacement of the fiber protein in the Ad capsid with a chimeric molecule containing a heterologous trimerization motif and a receptor-binding ligand. Our approach, which capitalized upon the overall structural similarity between the human Ad type 5 (Ad5) fiber and bacteriophage T4 fibritin proteins, has resulted in the generation of a genetically modified Ad5 incorporating chimeric fiber-fibritin proteins targeted to artificial receptor molecules. Gene transfer studies employing this novel viral vector have demonstrated its capacity to efficiently deliver a transgene payload to the target cells in a receptor-specific manner.
Adenovirus Hexon Protein Is a Potent Adjuvant for Activation of a Cellular Immune Response
Authors: Molinier-Frenkel Valérie , Lengagne Renée , Gaden Florence , Hong Saw-See , Choppin Jeannine , Gahery-Ségard Hanne , Boulanger Pierre , Guillet Jean-Gérard
Published: July 2002

The capacity of recombinant adenoviruses (rAd) to induce immunization against their transgene products has been well documented. In the present study, we evaluated the vaccinal adjuvant role of rAd independently of its vector function. BALB/c mice received one subcutaneous injection of a mixture of six lipopeptides (LP6) used as a model immunogen, along with AdE1° (109particles), a first-generation rAd empty vector. Although coinjected with a suboptimal dose of lipopeptides, AdE1° significantly improved the effectiveness of the vaccination, even in the absence of booster immunization. In contrast to mice that received LP6 alone or LP6 plus a mock adjuvant, mice injected with AdE1° plus LP6 developed both a polyspecific T-helper type 1 response and an effector CD8 T-cell response specific to at least two class I-restricted epitopes. The helper response was still observed when immunization was performed using LP6 plus a mixture of soluble capsid components released from detergent-disrupted virions. When mice were immunized with LP6 and each individual capsid component, i.e., hexon, penton base, or fiber, the results obtained suggested that hexon protein was responsible for the adjuvant effect exerted by disrupted Ad particles on the helper response to the immunogen. Our results thus have some important implications not only in vaccinology but also for gene therapy using rAd vectors.
Redox Regulation of Adenovirus-Induced AP-1 Activation by Overexpression of Manganese-Containing Superoxide Dismutase
Authors: Zhang Hannah J. , Drake Victoria J. , Xu Linjing , Hu Jianfang , Domann Frederick E. , Oberley Larry W. , Kregel Kevin C.
Published: July 2002

Adenovirus gene therapy is a promising tool in the clinical treatment of many genetic and acquired diseases. However, it has also caused pathogenic effects in organs such as the liver. The redox-sensitive transcription factors AP-1 and NF-κB have been implicated in these effects. To study the mechanisms of adenovirus-mediated AP-1 and NF-κB activation and the possible involvement of oxidative stress in adenovirus transduction, rats were injected with either replication-defective recombinant adenovirus with DNA containing the cytomegalovirus promoter region only (AdCMV), adenovirus containing human manganese-containing superoxide dismutase (MnSOD) cDNA (AdMnSOD), or vehicle. Compared to vehicle and AdCMV transduction, MnSOD gene transfer yielded a fivefold increase in liver MnSOD activity 7 days postinjection. Gel shift assay showed that AdCMV transduction induced DNA binding activity for AP-1 but not NF-κB. MnSOD overexpression abolished this activation. Western blotting analysis of c-Fos and c-Jun suggested that up-regulation of c-
fos
and c-
jun
gene expression does not directly contribute to the induction of AP-1 activation. Glutathione/glutathione disulfide ratios were decreased by adenovirus transduction and restored by MnSOD overexpression. The AP-1 binding activity that was induced by AdCMV was decreased by immunoprecipitation of Ref-1 protein. Ref-1 involvement was confirmed by restoration of AP-1 binding activity after the immunoprecipitated Ref-1 protein had been added back. AP-1 DNA binding activity was also elevated in control and AdMnSOD-injected rats after addition of the immunoprecipitated Ref-1 protein. These data indicate that cellular transduction by recombinant adenovirus stimulates AP-1 DNA binding activity. Furthermore, our results suggest that MnSOD overexpression decreases AP-1 DNA binding activity by regulating intracellular redox status, with the possible involvement of Ref-1 in this redox-sensitive pathway.
Role of Regulatory Elements and the MAPK/ERK or p38 MAPK Pathways for Activation of Human Cytomegalovirus Gene Expression
Authors: Chen Jiping , Stinski Mark F.
Published: July 2002

A series of recombinant viruses with either site-specific mutations or various deletions of the early UL4 promoter of human cytomegalovirus were used to determine the roles of regulatory elements and the effects of the mitogen-activated protein kinase (MAPK) pathways. Viral gene expression was regulated by upstream
cis
-acting sites and by basic promoter elements that respond to the MAPK signal transduction pathways. Inhibitors of either the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway or the p38 MAPK pathway affected expression equally with either wild-type or mutant early UL4 promoters in the viral genome, indicating that the effects of the inhibitors are not exclusive for a single transcription factor. The minimal responsive element is the TATA box-containing early viral promoter.
Kaposi's Sarcoma-Associated Herpesvirus K3 Utilizes the Ubiquitin-Proteasome System in Routing Class I Major Histocompatibility Complexes to Late Endocytic Compartments
Authors: Lorenzo Mayra E. , Jung Jae U. , Ploegh Hidde L.
Published: July 2002

Human herpesvirus 8 (HHV8) downregulates major histocompatibility complex (MHC) class I complexes from the plasma membrane via two of its genes, K3 and K5. The N termini of K3 and K5 contain a plant homeodomain (PHD) predicted to be structurally similar to RING domains found in E3 ubiquitin ligases. In view of the importance of the ubiquitin-proteasome system in sorting within the endocytic pathway, we analyzed its role in downregulation of MHC class I complexes in cells expressing K3. Proteasome inhibitors as well as cysteine and aspartyl protease inhibitors stabilize MHC class I complexes in cells expressing K3. However, proteasome inhibitors differentially affect sorting of MHC class I complexes within the endocytic pathway and prevent their delivery to a dense endosomal compartment. In this compartment, the cytoplasmic tail of MHC class I complexes is cleaved by cysteine proteases. The complex is then cleaved within the plane of the membrane by an aspartyl protease, resulting in a soluble MHC class I fragment composed of the lumenal domain of the heavy chain, β2-microglobulin (β2m), and peptide. We conclude that K3 not only directs internalization, but also targets MHC class I complexes to a dense endocytic compartment on the way to lysosomes in a ubiquitin-proteasome-dependent manner.
Langat Flavivirus Protease NS3 Binds Caspase-8 and Induces Apoptosis
Authors: Prikhod'ko Grigori G. , Prikhod'ko Elena A. , Pletnev Alexander G. , Cohen Jeffrey I.
Published: July 2002

The flavivirus NS3 protein plays an important role in the cleavage and processing of the viral polyprotein and in the synthesis of the viral RNA. NS3 recruits NS2B and NS5 proteins to form complexes possessing protease and replicase activities through protease and nucleoside triphosphatase/helicase domains. We have found that NS3 also induces apoptosis. Expression of the Langat (LGT) virus NS3 protein resulted in a cleavage of cellular DNA and reduced the viability of cells. Coexpression of NS3 with apoptotic inhibitors (CrmA and P35) and addition of caspase peptide substrates (Z-VAD-FMK and Z-IETD-FMK) toNS3-transfected cells blocked NS3-induced apoptosis. In cotransfection experiments, NS3 bound to caspase-8 and enhanced caspase-8-mediated apoptosis. NS3 and caspase-8 colocalized in the cytoplasm of transfected cells. Deletion analysis demonstrated that at least two regions of NS3 contribute to its apoptotic activities. The protease and helicase domains are each able to bind to caspase-8, while the protease domain alone induces apoptosis. The protease domain and tetrahelix region of the helicase domain are required for NS3 to augment caspase-8-mediated apoptosis. Thus, the LGT virus NS3 protein is a multifunctional protein that binds to caspase-8 and induces apoptosis.
RelB-p50 NF-κB Complexes Are Selectively Induced by Cytomegalovirus Immediate-Early Protein 1: Differential Regulation of Bcl-xLPromoter Activity by NF-κB Family Members
Authors: Jiang H. Y. , Petrovas Constantinos , Sonenshein Gail E.
Published: July 2002

The NF-κB/Rel family has been implicated in control of transcription of the Bcl-xLgene, a target which mediates cell survival signals. The cytomegalovirus (CMV) immediate-early protein 1 (IE1) was previously shown to induce NF-κB activity. Here, we report that in both vascular smooth muscle cells (SMCs) and NIH 3T3 cells, surprisingly, IE1 failed to induce Bcl-xLpromoter activity, although it induced activity of E8-CAT, a reporter construct driven by two copies of the NF-κB element upstream of the c-mycpromoter (upstream regulatory element [URE]). Thus, the subunit nature of the NF-κB/Rel factors induced by IE1 was examined using immunofluorescence and immunoblotting. IE1 was found to selectively induce nuclear RelB and p50 in SMCs and NIH 3T3 cells. An increase in RelB protein mediated by IE1 could, in part, be related to an increase in steady-staterelBmRNA levels. Consistent with this subunit identification, IE1 was unable to induce E8-CAT activity inrelB−/−murine embryonic fibroblast cells. In cotransfection analysis of SMCs and NIH 3T3 cells, RelB and p50 proteins failed to induce Bcl-xLpromoter activity while inducing E8-CAT. Furthermore, the NF-κB element of the Bcl-xLpromoter only weakly bound RelB-p50 complexes compared to the URE NF-κB element. Overall, these findings demonstrate in SMCs and NIH 3T3 cells that the CMV IE1 protein selectively induces RelB and p50, which fail to activate the Bcl-xLpromoter, indicating a strong specificity of binding and activity for the RelB member of the NF-κB family. Furthermore, our results implicate RelB in CMV infection of cells such as vascular SMCs.
Visualization of Polydnavirus Sequences in a Parasitoid Wasp Chromosome
Authors: Belle Elise , Beckage Nancy E. , Rousselet Jérôme , Poirié Marylène , Lemeunier Françoise , Drezen Jean-Michel
Published: July 2002

Polydnaviruses, obligatorily associated with endoparasitoid wasps, are unique in that their segmented genome is composed of multiple double-stranded DNA circles. We present here the first cytological evidence that virus segments are integrated in the wasp genome, obtained by using in situ hybridization of virus probes with viral sequences in the chromosomes of a wasp from the braconid family of hymenopterans.
Previously Infected and Recovered Chimpanzees Exhibit Rapid Responses That Control Hepatitis C Virus Replication upon Rechallenge
Authors: Major Marian E. , Mihalik Kathleen , Puig Montserrat , Rehermann Barbara , Nascimbeni Michelina , Rice Charles M. , Feinstone Stephen M.
Published: July 2002

Responses in three chimpanzees were compared following challenge with a clonal hepatitis C virus (HCV) contained in plasma from an animal that had received infectious RNA transcripts. Two of the chimpanzees (Ch1552 and ChX0186) had recovered from a previous infection with HCV, while the third (Ch1605) was a naïve animal. All animals were challenged by reverse titration with decreasing dilutions of plasma and became serum RNA positive following challenge. Ch1605 displayed a typical disease profile for a chimpanzee. We observed increasing levels of serum RNA from week 1 postinoculation (p.i.), reaching a peak of 10
6
copies/ml at week 9 p.i., and alanine aminotransferase (ALT) elevations and seroconversion to HCV antibodies at week 10 p.i. In contrast, both Ch1552 and ChX0186 exhibited much shorter periods of viremia (4 weeks), low serum RNA levels (peak, 10
3
copies/ml), and minimal ALT elevations. A comparison of intrahepatic cytokine levels in Ch1552 and Ch1605 showed greater and earlier gamma interferon (IFN-γ) and tumor necrosis factor alpha responses in the previously infected animal, responses that were 30-fold greater than baseline responses at week 4 p.i. for IFN-γ in Ch1552 compared to 12-fold in Ch1605 at week 10 p.i. These data indicate (i) that clonal HCV generated from an infectious RNA transcript will lead to a typical HCV infection in naïve chimpanzees, (ii) that there are memory immune responses in recovered chimpanzees that control HCV infection upon rechallenge, and (iii) that these responses seem to be T-cell mediated, as none of the animals had detectable antibody against the HCV envelope glycoproteins. These observations have encouraging implications for the development of a vaccine for HCV.
Antibody-Mediated Enhancement of Human Immunodeficiency Virus Type 1 Infectivity Is Determined by the Structure of gp120 and Depends on Modulation of the gp120-CCR5 Interaction
Authors: Guillon Christophe , Schutten Martin , Boers Patrick H. M. , Gruters Rob A. , Osterhaus Albert D. M. E.
Published: July 2002

In this study, we characterized the viral determinants of coreceptor usage in relation to susceptibility to antibody-mediated neutralization or enhancement of infectivity by using chimeras of three highly related human immunodeficiency virus type 1 (HIV-1) isolates of different phenotypes. We found that the V3 region was the main determinant of antibody-mediated enhancement and coreceptor specificity but that the overall structure of gp120 was also important for these properties. Constructs susceptible to antibody-mediated enhancement preferentially use CCR5 as a coreceptor, in contrast to constructs that were neutralized or not affected. Using monoclonal antibodies directed against CD4 or CCR5, we were able to show that antibody-mediated enhancement was CD4 dependent. Altogether, our results suggest that the modulation of the interaction of gp120 with CCR5 is the mechanism underlying antibody-mediated enhancement of HIV-1 infectivity.
Both Viral Transcription and Replication Are Reduced when the Rabies Virus Nucleoprotein Is Not Phosphorylated
Authors: Wu Xianfu , Gong Xiaoming , Foley Heather D. , Schnell Matthias J. , Fu Zhen F.
Published: July 2002

Rabies virus nucleoprotein (N) plays vital roles in regulation of viral RNA transcription and replication by encapsidation of the nascent genomic RNA. Rabies virus N is phosphorylated, and previous studies demonstrated that mutation of the phosphorylated serine at position 389 to alanine resulted in reduction of viral transcription and/or replication of a rabies virus minigenome. In the present study, we mutated the serine (S) at position 389 to alanine (A), glycine (G), aspartic acid (D), asparagine (N), glutamic acid (E), and glutamine (Q) and examined the effects of these mutations on rabies virus transcription and replication in the minigenome. Furthermore, mutations from S to A, S to D, and S to E were also incorporated into the full-length infectious virus. Mutation of the serine to each of the other amino acids resulted in the synthesis of an unphosphorylated N and reduction of viral transcription and replication in the minigenome. Mutations from S to A and S to D also resulted in reduction of both viral transcription and replication in full-length infectious viruses. Growth curve studies indicated that production of the mutant virus with the S-to-A mutation (L16A) was as much as 10,000-fold less than that of the wild-type virus (L16). Northern blot hybridization with rabies virus gene probes revealed that the rates of viral transcription and replication were reduced by as much as 10-fold in the mutant viruses when the N was not phosphorylated. Interpretation of the data from the minigenome system and the full-length infectious virus indicates that phosphorylation of rabies virus N is necessary for replication. Further studies involving cycloheximide treatment of infected cells revealed that viral transcription was also reduced when the N was not phosphorylated. Taken together, these results provide definitive evidence that N phosphorylation plays an important role in the processes of rabies virus transcription and replication.
Modified Apparatus for Measuring V-L Equilibria by Static Method
Authors: Surový Július , Graczová Elena , Bafrncová Soňa
Published: July 2002

A modified static apparatus for measuring the vapour liquid equilibrium data is described. It is a modification of the apparatus originally proposed by Surovy and Dojcansky (1974). The construction alterations applied to the apparatus improve and simplify its functioning. It is checked on measuring three binary systems: benzene - n-heptane, cyclohexane - cyclohexanone and benzene - 2-propanol. The agreement of the experimental and tabulated data proves the suitability of the apparatus for obtaining the V-L equilibrium data.
The Effect of Extra-Framework Aluminum in Dealuminated ZSM-5 Zeolites on the Transformation of Aromatic Hydrocarbons
Authors: Čejka Jiří , Vondrová Alena , Wichterlová Blanka , Jerschkewitz Hans G. , Lischke Günter , Schreier Ellen
Published: July 2002

H-ZSM-5 zeolite was dealuminated by steaming under "mild" and "severe" conditions at 675 and 975 K for 71.5 and 0.5 h, respectively. It resulted in a comparable number of Al atoms released from the zeolite framework, although of their different nature. The "mild" zeolite steaming yielded Al species easily removable from the zeolite by an acid leaching, in contrast to the zeolite steamed at "severe" conditions. The extra-framework Al species contributed to both a substantially higher para-selectivity in toluene disproportionation and its alkylation with ethylene and to an increased rate of side reactions leading to the zeolite deactivation by coke formation in toluene alkylation with ethylene.
Gould-Jacobs Reaction of 5- and 6-Amino-2-substitutedBenzoxazoles. III. Reaction with 3-Ethoxy-2-cyanopropenonitrile and Ethyl 3-Ethoxy-2-cyanopropenoate
Authors: Heleyová Katarína , Ilavský Dušan
Published: July 2002

Nucleophilic reaction of 5-amino- (1) and 6-amino-2-substituted benzoxazoles (2) with 3-ethoxy-2-cyanopropenonitrile (3) afforded the respective benzoxazolylaminomethylenemalononitriles 5 and 6. The amino derivatives 1 and 2 reacted with ethyl 3-ethoxy-2-cyanopropenoate (4) to give the corresponding esters of benzoxazolylamino-2-cyanopropenoic acid 7 and 8, respectively. The products 7 on thermal cyclization at 250-260 °C in a mixture of diphenyl ether and biphenyl afforded a mixture of angularly and linearly annelated 5-nitrile-4-oxooxazolo[4,5-f]quinoline 9 and 7-nitrile-8-oxooxazolo[5,4-g]quinoline 10; under the same conditions compounds 8 were converted into 8-nitrile-9-oxooxazolo[5,4-f]quinolines 11 and 6-nitrile-5-oxooxazolo[4,5-g]quinolines 12.