bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mapping and Characterization of the N-Terminal I Domain of Human Immunodeficiency Virus Type 1 Pr55
Gag
Authors: Sandefur Stephanie , Smith Rita M. , Varthakavi Vasundhara , Spearman Paul 
Published: July 2002

ABSTRACT

Human immunodeficiency virus (HIV) type 1 particles assemble at the plasma membrane of cells in a manner similar to that of the type C oncoretroviruses. The Pr55
Gag
molecule directs the assembly process and is sufficient for particle assembly in the absence of all other viral gene products. The I domain is an assembly domain that has been previously localized to the nucleocapsid (NC) region of Gag. In this study we utilized a series of Gag-green fluorescent protein (GFP) fusion proteins to precisely identify sequences that constitute the N-terminal I domain of Pr55
Gag
. The minimal sequence required for the I domain was localized to the extreme N terminus of NC. Two basic residues (arginine 380 and arginine 384) within the initial seven residues of NC were found to be critical for the function of the N-terminal I domain. The presence of positive charge alone in these two positions, however, was not sufficient to mediate the formation of dense Gag particles. The I domain was required for the formation of detergent-resistant complexes of Gag protein, and confocal microscopy demonstrated that the I domain was also required for the formation of punctate foci of Gag proteins at the plasma membrane. Electron microscopic analysis of cells expressing Gag-GFP fusion constructs with an intact I domain revealed numerous retrovirus-like particles (RVLPs) budding from the plasma membrane, while I domain-deficient constructs failed to generate visible RVLPs. These results provide evidence that Gag-Gag interactions mediated by the I domain play a central role in the assembly of HIV particles.
Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism
Authors: Wu Pei , Xiao Wu , Conlon Thomas , Hughes Jeffrey , Agbandje-McKenna Mavis , Ferkol Thomas , Flotte Terence , Muzyczka Nicholas 
Published: July 2002

ABSTRACT
Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be β-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.
Persistent Virus Infection despite Chronic Cytotoxic T-Lymphocyte Activation in Gamma Interferon-Deficient Mice Infected with Lymphocytic Choriomeningitis Virus
Authors: Bartholdy Christina , Christensen Jan Pravsgaard , Wodarz Dominik , Thomsen Allan Randrup 
Published: July 2002

ABSTRACTThe role of gamma interferon (IFN-γ) in the permanent control of infection with a noncytopathic virus was studied by comparing immune responses in wild-type and IFN-γ-deficient (IFN-γ −/−) mice infected with a slowly invasive strain of lymphocytic choriomeningitis virus (LCMV Armstrong). While wild-type mice rapidly cleared the infection, IFN-γ −/− mice became chronically infected. Virus persistence in the latter mice did not reflect failure to generate cytotoxic T-lymphocyte (CTL) effectors, as an unimpaired primary CTL response was observed. Furthermore, while ex vivo CTL activity gradually declined in wild-type mice, long-standing cytolytic activity was demonstrated in IFN-γ −/− mice. The prolonged effector phase in infected IFN-γ −/− mice was associated with elevated numbers of CD8+T cells. Moreover, a higher proportion of these cells retained an activated phenotype and was actively cycling. However, despite the increased CD8+T-cell turnover, which might have resulted in depletion of the memory CTL precursor pool, no evidence for exhaustion was observed. In fact, at 3 months postinfection we detected higher numbers of LCMV-specific CTL precursors in IFN-γ −/− mice than in wild-type mice. These findings indicate that in the absence of IFN-γ, CTLs cannot clear the infection and are kept permanently activated by the continuous presence of live virus, resulting in a delicate new balance between viral load and immunity. This interpretation of our findings is supported by mathematical modeling describing the effect of eliminating IFN-γ-mediated antiviral activity on the dynamics between virus replication and CTL activity.
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

ABSTRACTIn 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

ABSTRACT

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

ABSTRACT

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

ABSTRACTPrevious 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

ABSTRACT
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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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

ABSTRACT
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

ABSTRACT
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.