bluuebunny/crossref_metadata_2025_split huggingface dataset:
Underrepresentation of the 3′ Region of the Capsid Pregenomic RNA of Duck Hepatitis B Virus
Authors: Ostrow Kristin M. , Loeb Daniel D. 
Published: February 2004

ABSTRACT
The pregenomic RNA (pgRNA) of hepadnaviruses is packaged into capsids where it is reverse transcribed to yield mature DNA genomes. This report describes differences between the 3′ region and other regions of the pgRNA isolated from capsids. Analysis of capsid pgRNA isolated by using an established method involving micrococcal nuclease treatment demonstrated reduced levels of the 3′ region of the pgRNA compared to the 5′ region. This underrepresentation of the 3′ region was partly a result of microccocal nuclease digestion of the 3′ region because isolation of capsid pgRNA by an alternative method that did not involve nuclease treatment led to a greater, but not complete, recovery of the 3′ region. These results indicate that the 3′ region of the capsid pgRNA is susceptible to micrococcal nuclease digestion during its isolation and that the 3′ region can still be underrepresented when capsid pgRNA is isolated without nuclease digestion. Additional experiments show that the 3′ ends of capsid pgRNA isolated by micrococcal nuclease treatment are heterogeneously dispersed from nucleotide 2577 to the poly(A) tail. These data provide evidence that the 3′ region of the capsid pgRNA has biochemical properties different from those of its 5′ region. Possibly, the 3′ region of the pgRNA is not packaged into the interior of the capsid but rather is associated with a part of the capsid where it is susceptible to microccocal nuclease digestion.
Transforming Growth Factor β Enhances the Glucocorticoid Response of the Mouse Mammary Tumor Virus Promoter through Smad and GA-Binding Proteins
Authors: Aurrekoetxea-Hernández Koldo , Buetti Elena 
Published: February 2004

ABSTRACTTissue-specific transcription is advantageously investigated by using viral promoters, which are selected for compact regulatory elements. Mouse mammary tumor virus (MMTV) has adapted to specialized cell types and targets initially B lymphocytes. We previously showed that, in B-cell lines, glucocorticoid-induced MMTV transcription requires an ETS family factor, GA-binding protein (GABP), bound in tandem to the MMTV DNA next to the glucocorticoid receptor (GR). We now report that transforming growth factor β (TGF-β) superinduces this response up to 10-fold through binding of its effectors, Smads, between the GABP-binding motifs. The basal level was unaffected. The TGF-β-glucocorticoid cooperation also depended on GR and GABP binding, was transferable to another promoter, and occurred both with transiently transfected and with integrated templates. Smad3 associated in vitro with GR, with GABPα (via the MH2 domain), and with GABPβ, Smad4 only with GABPα. Interactions of Smad3 with GABP (when coexpressed or endogenous to B cells) were shown by coprecipitation and by mammalian two-hybrid assay. This composite DNA element integrates three signaling pathways deriving from TGF-β, glucocorticoid hormones, and a unique ETS factor, and may allow MMTV to exploit factors from the milk. It may as well indicate novel possibilities for cellular regulatory networks.
Protection against Mucosal Simian Immunodeficiency Virus SIV
mac251
Challenge by Using Replicating Adenovirus-SIV Multigene Vaccine Priming and Subunit Boosting
Authors: Patterson L. Jean , Malkevitch Nina , Venzon David , Pinczewski Joel , Gómez-Román Victor Raúl , Wang Liqun , Kalyanaraman V. S. , Markham Phillip D. , Robey Frank A. , Robert-Guroff Marjorie 
Published: February 2004

ABSTRACT

Whereas several recent AIDS vaccine strategies have protected rhesus macaques against a pathogenic simian/human immunodeficiency virus (SHIV)
89.6P
challenge, similar approaches have provided only modest, transient reductions in viral burden after challenge with virulent, pathogenic SIV, which is more representative of HIV infection of people. We show here that priming with replicating adenovirus recombinants encoding SIV
env/rev
,
gag
, and/or
nef
genes, followed by boosting with SIV gp120 or an SIV polypeptide mimicking the CD4 binding region of the envelope, protects rhesus macaques from intrarectal infection with the highly pathogenic SIV
mac251
. Using trend analysis, significant reductions in acute-phase and set point viremia were correlated with anti-gp120 antibody and cellular immune responses, respectively. Within immunization groups exhibiting significant protection, a subset (39%) of macaques have exhibited either no viremia, cleared viremia, or controlled viremia at the threshold of detection, now more than 40 weeks postchallenge. This combination prime-boost strategy, utilizing replication competent adenovirus, is a promising alternative for HIV vaccine development.
Specificity of Interaction of INI1/hSNF5 with Retroviral Integrases and Its Functional Significance
Authors: Yung Eric , Sorin Masha , Wang Emilie-Jeanne , Perumal Seena , Ott David , Kalpana Ganjam V. 
Published: February 2004

ABSTRACT
Integrase interactor 1 (INI1)/hSNF5 is a host factor that directly interacts with human immunodeficiency virus type 1 (HIV-1) integrase and is incorporated into HIV-1 virions. Here, we show that while INI1/hSNF5 is completely absent from purified microvesicular fractions, it is specifically incorporated into HIV-1 virions with an integrase-to-INI1/hSNF5 stoichiometry of approximately 2:1 (molar ratio). In addition, we show that INI1/hSNF5 is not incorporated into related primate lentiviral and murine retroviral particles despite the abundance of the protein in producer cells. We have found that the specificity in incorporation of INI1/hSNF5 into HIV-1 virions is directly correlated with its ability to exclusively interact with HIV-1 integrase but not with other retroviral integrases. This specificity is also reflected in our finding that the transdominant mutant S6, harboring the minimal integrase interaction domain of INI1/hSNF5, blocks HIV-1 particle production but not that of the other retroviruses in 293T cells. Taken together, these results suggest that INI1/hNSF5 is a host factor restricted for HIV-1 and that S6 acts as a highly specific and potent inhibitor of HIV-1 replication.
Genetic Basis of Hypersusceptibility to Protease Inhibitors and Low Replicative Capacity of Human Immunodeficiency Virus Type 1 Strains in Primary Infection
Authors: Leigh Brown Andrew J. , Frost Simon D. W. , Good Benjamin , Daar Eric S. , Simon Viviana , Markowitz Martin , Collier Ann C. , Connick Elizabeth , Conway Brian , Margolick Joseph B. , Routy Jean-Pierre , Corbeil Jacques , Hellmann Nicholas S. , Richman Douglas D. , Little Susan J. 
Published: February 2004

ABSTRACTThe initial virus strains from as many as 12% of individuals with primary human immunodeficiency virus (HIV) infection have a 50% inhibitory concentration ≤0.4-fold that of HIV type 1NL4-3(HIV-1NL4-3) to ritonavir (hypersusceptibility [HS]). There is also substantial variation in replicative capacity (RC) or an in vitro assay of the contributions of protease (PR) and reverse transcriptase to viral fitness. In chronically infected antiretrovirally treated patients, amprenavir HS has been associated with the mutation N88S in PR, but this mutation is not seen in untreated patients. In this study, virus strains from 182 cases of primary HIV infection were analyzed, and a highly significant association between HS and low RC (≤10% that of HIV-1NL4-3) was observed (P< 10−6). Multivariate analysis was used to determine the genotypic basis of ritonavir HS, analyzing all polymorphic amino acid sites and insertions from p7gag through PR. Decision tree models developed on the entire Gag-plus-PR data set and on PR alone gave overall correct classifications of 73 and 72%, respectively, on cross-validation. They were also able to predict low RC, with sensitivities of 69 and 62% and specificities of 84 and 70%, respectively. The analysis shows that ritonavir HS in untreated primary HIV infection is not associated with single mutations but with combinations of amino acids at polymorphic sites and that the same genotypes which confer HS to PR inhibitors confer low RC. This supports the view that variation in PR function is directly responsible for variation in fitness among strains in primary infection.
Underwhelming the Immune Response: Effect of Slow Virus Growth on CD8+-T-Lymphocyte Responses
Authors: Bocharov Gennady , Ludewig Burkhard , Bertoletti Antonio , Klenerman Paul , Junt Tobias , Krebs Philippe , Luzyanina Tatyana , Fraser Cristophe , Anderson Roy M. 
Published: February 2004

ABSTRACTThe speed of virus replication has typically been seen as an advantage for a virus in overcoming the ability of the immune system to control its population growth. Under some circumstances, the converse may also be true: more slowly replicating viruses may evoke weaker cellular immune responses and therefore enhance their likelihood of persistence. Using the model of lymphocytic choriomeningitis virus (LCMV) infection in mice, we provide evidence that slowly replicating strains induce weaker cytotoxic-T-lymphocyte (CTL) responses than a more rapidly replicating strain. Conceptually, we show a “bell-shaped” relationship between the LCMV growth rate and the peak CTL response. Quantitative analysis of human hepatitis C virus infections suggests that a reduction in virus growth rate between patients during the incubation period is associated with a spectrum of disease outcomes, from fulminant hepatitis at the highest rate of viral replication through acute resolving to chronic persistence at the lowest rate. A mathematical model for virus-CTL population dynamics (analogous to predator [CTL]-prey [virus] interactions) is applied in the clinical data-driven analysis of acute hepatitis B virus infection. The speed of viral replication, through its stimulus of host CTL responses, represents an important factor influencing the pathogenesis and duration of virus persistence within the human host. Viruses with lower growth rates may persist in the host because they “sneak through” immune surveillance.
Expansion of Protective CD8+T-Cell Responses Driven by Recombinant Cytomegaloviruses
Authors: Karrer Urs , Wagner Markus , Sierro Sophie , Oxenius Annette , Hengel Hartmut , Dumrese Tilman , Freigang Stefan , Koszinowski Ulrich H. , Phillips Rodney E. , Klenerman Paul 
Published: February 2004

ABSTRACTCD8+T cells are critical for the control of many persistent viral infections, such as human immunodeficiency virus, hepatitis C virus, Epstein-Barr virus, and cytomegalovirus (CMV). In most infections, large CD8+-T-cell populations are induced early but then contract and are maintained thereafter at lower levels. In contrast, CD8+T cells specific for murine CMV (MCMV) have been shown to gradually accumulate after resolution of primary infection. This unique behavior is restricted to certain epitopes, including an immunodominant epitope derived from the immediate-early 1 (IE1) gene product. To explore the mechanism behind this further, we measured CD8+-T-cell-mediated immunity induced by recombinant MCMV-expressing epitopes derived from influenza A virus or lymphocytic choriomeningitis virus placed under the control of an IE promoter. We observed that virus-specific CD8+-T-cell populations were induced and that these expanded gradually over time. Importantly, these CD8+T cells provided long-term protection against challenge without boosting. These results demonstrate a unique pattern of accumulating T cells, which provide long-lasting immune protection, that is independent of the initial immunodominance of the epitope and indicates the potential of T-cell-inducing vaccines based on persistent vectors.
Promoting Trimerization of Soluble Human Immunodeficiency Virus Type 1 (HIV-1) Env through the Use of HIV-1/Simian Immunodeficiency Virus Chimeras
Authors: Center Rob J. , Lebowitz Jacob , Leapman Richard D. , Moss Bernard 
Published: February 2004

ABSTRACT
The envelope proteins (Env) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) form homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained, but is less stable, after cleavage in a Golgi compartment and transport to the surface of infected cells. Functional, virion-associated HIV-1 and SIV Env have an almost exclusively trimeric structure. In addition, a soluble form of SIV Env (gp140) forms a nearly homogeneous population of trimers. Here, we describe the oligomeric structure of soluble, uncleaved HIV-1 gp140 and modifications that promote a stable trimeric structure. Biochemical and biophysical analyses, including sedimentation equilibrium and scanning transmission electron microscopy, revealed that unmodified HIV-1 gp140 purified as a heterogeneous range of oligomeric species, including dimers and aggregates. Deletion of the V2 domain alone or, especially, both the V1 and V2 domains reduced dimer formation but promoted aggregation rather than trimerization. Expressing gp140 with mannose-only oligosaccharides did not eliminate heterogeneity. Replacement of the entire gp41 segment of HIV-1 gp140 or just the N-terminal half (85 amino acids) of this segment with the corresponding region of SIV was sufficient to confer efficient trimerization for gp140 derived from clade B and C isolates. Importantly, the relatively small segment of the HIV Env replaced by SIV sequences contains no known targets of neutralizing antibody. The soluble trimeric form of HIV-1 Env should prove useful for assessment of antigenic structure and immunogenicity.
Role for CCR5Δ32 Protein in Resistance to R5, R5X4, and X4 Human Immunodeficiency Virus Type 1 in Primary CD4+Cells
Authors: Agrawal Lokesh , Lu Xihua , Qingwen Jin , VanHorn-Ali Zainab , Nicolescu Ioan Vlad , McDermott David H. , Murphy Philip M. , Alkhatib Ghalib 
Published: February 2004

ABSTRACTCCR5Δ32is a loss-of-function mutation that abolishes cell surface expression of the human immunodeficiency virus (HIV) coreceptor CCR5 and provides genetic resistance to HIV infection and disease progression. Since CXCR4 and other HIV coreceptors also exist, we hypothesized thatCCR5Δ32-mediated resistance may be due not only to the loss of CCR5 function but also to a gain-of-function mechanism, specifically the active inhibition of alternative coreceptors by the mutant CCR5Δ32 protein. Here we demonstrate that efficient expression of the CCR5Δ32 protein in primary CD4+cells by use of a recombinant adenovirus (Ad5/Δ32) was able to down-regulate surface expression of both wild-type CCR5 and CXCR4 and to confer broad resistance to R5, R5X4, and X4 HIV type 1 (HIV-1). This may be important clinically, since we found that CD4+cells purified from peripheral blood mononuclear cells of individuals who were homozygous forCCR5Δ32, which expressed the mutant protein endogenously, consistently expressed lower levels of CXCR4 and showed less susceptibility to X4 HIV-1 isolates than cells from individuals lacking the mutation. Moreover, CD4+cells from individuals who were homozygous forCCR5Δ32expressed the mutant protein in five of five HIV-exposed, uninfected donors tested but not in either of two HIV-infected donors tested. The mechanism of inhibition may involve direct scavenging, since we were able to observe a direct interaction of CCR5 and CXCR4 with CCR5Δ32, both by genetic criteria using the yeast two-hybrid system and by biochemical criteria using the coimmunoprecipitation of heterodimers. Thus, these results suggest that at least two distinct mechanisms may account for genetic resistance to HIV conferred byCCR5Δ32: the loss of wild-type CCR5 surface expression and the generation of CCR5Δ32 protein, which functions as a scavenger of both CCR5 and CXCR4.
A Cysteine-Rich Plant Protein Potentiates
Potyvirus
Movement through an Interaction with the Virus Genome-Linked Protein VPg
Authors: Dunoyer P. , Thomas C. , Harrison S. , Revers F. , Maule A. 
Published: February 2004

ABSTRACT

We have identified a cellular factor that interacts with the virus genome-linked proteins (VPgs) of a diverse range of potyviruses. The factor, called
Potyvirus
VPg-interacting protein (PVIP), is a plant-specific protein with homologues in all the species examined, i.e., pea,
Arabidopsis thaliana
, and
Nicotiana benthamiana
. The sequence of PVIP does not identify a specific function, although the existence of a “PHD finger” domain may implicate the protein in transcriptional control through chromatin remodeling. Deletion analysis using the yeast two-hybrid system showed that the determinants of the interaction lay close to the N terminus of VPg; indeed, the N-terminal 16 amino acids were shown to be both necessary and sufficient for the interaction with at least one PVIP protein. From a sequence comparison of different potyvirus VPg proteins, a specific amino acid at position 12 was directly implicated in the interaction. This part of VPg is distinct from regions associated with other functional roles of VPg. Through mutation of
Turnip mosaic virus
(TuMV) at VPg position 12, we showed that the interaction with PVIP affected systemic symptoms in infected plants. This resulted from reduced cell-to-cell and systemic movement more than reduced virus replication, as visualized by comparing green fluorescent protein-tagged wild-type and mutant viruses. Furthermore, by using RNA interference of PVIP in
Arabidopsis
, we showed that reduced expression of
PVIP
genes reduced susceptibility to TuMV infection. We conclude that PVIP functions as an ancillary factor to support potyvirus movement in plants.
Characterization of Rotavirus Cell Entry
Authors: Sánchez-San Martín Claudia , López Tomás , Arias Carlos F. , López Susana 
Published: February 2004

ABSTRACT
While recently we have learned much about the viral and cellular proteins involved in the initial attachment of rotaviruses to MA104 cells, the mechanism by which these viruses reach the interior of the cell is poorly understood. For this study, we observed the effects of drugs and of dominant-negative mutants, known to impair clathrin-mediated endocytosis and endocytosis mediated by caveolae, on rotavirus cell infection. Rotaviruses were able to enter cells in the presence of compounds that inhibit clathrin-mediated endocytosis as well as cells overexpressing a dominant-negative form of Eps15, a protein crucial for the assembly of clathrin coats. We also found that rotaviruses infected cells in which caveolar uptake was blocked; treatment with the cholesterol binding agents nystatin and filipin, as well as transfection of cells with dominant-negative caveolin-1 and caveolin-3 mutants, had no effect on rotavirus infection. Interestingly, cells treated with methyl-β-cyclodextrin, a drug that sequesters cholesterol from membranes, and cells expressing a dominant-negative mutant of the large GTPase dynamin, which is known to function in several membrane scission events, were not infected by rotaviruses, indicating that cholesterol and dynamin play a role in the entry of rotaviruses.
Role of Human Immunodeficiency Virus Type 1 Matrix Phosphorylation in an Early Postentry Step of Virus Replication
Authors: Kaushik Rajnish , Ratner Lee 
Published: February 2004

ABSTRACT
The matrix domain (MA) is important for targeting of human immunodeficiency virus type 1 Gag assembly to the plasma membrane, envelope incorporation into virions, preintegration complex import into the nucleus, and nuclear export of viral RNA. Myristylation and phosphorylation are key regulatory events for MA function. Previous studies have indicated that MA phosphorylation at serine (Ser) residues is important for viral replication. This study defines the molecular mechanisms of virus particle assembly and infectivity through a detailed study of the role of MA serine phosphorylation. We show that the combined mutation of Ser residues at positions 9, 67, 72, and 77 impairs viral infectivity in dividing and nondividing cells, although the assembly of these Ser mutant viruses is comparable to that of wild-type virus. This defect can be rescued by pseudotyping these mutant viruses with vesicular stomatitis virus G protein, suggesting that these serine residues are critical in an early postentry step of viral infection. The phosphorylation level of MA in defective mutant viruses was severely reduced compared to that of the wild type, suggesting that phosphorylation of Ser-9, -67, -72, and -77 is important for an early postentry step during virus infection.
Cell Cycle Requirements for Transduction by Foamy Virus Vectors Compared to Those of Oncovirus and Lentivirus Vectors
Authors: Trobridge Grant , Russell David W. 
Published: February 2004

ABSTRACT

Retroviral vectors based on foamy viruses (FV) are efficient gene delivery vehicles for therapeutic and research applications. While previous studies have shown that FV vectors transduce quiescent cell cultures more efficiently than oncoviral vectors, their specific cell cycle requirements have not been determined. Here we compare the transduction frequencies of FV vectors with those of onco- and lentiviral vectors in nondividing and dividing normal human fibroblasts by several methods. FV vectors transduced serum-deprived fibroblast cultures more efficiently than oncoretroviral vectors and at rates comparable to those of lentiviral vectors. However, in these cultures FV vectors only transduced a subpopulation of proliferating cells, as determined by bromodeoxyuridine staining for DNA synthesis. In contrast to lentiviral vectors, FV vectors were unable to transduce human fibroblasts arrested by aphidicolin (G
1
/S phase) or γ-irradiation (G
2
phase), and a partial cell cycle that included mitosis but not DNA synthesis was required. We could not determine if mitosis facilitated nuclear entry of FV vectors, since cell-free vector preparations contained long terminal repeat circles, precluding their use as nuclear markers. In contrast to oncoviral vectors, both FV and lentiviral vectors efficiently transduced G
0
fibroblasts that were later stimulated to divide. In the case of FV vectors, this was due to the persistence of a stable transduction intermediate in quiescent cells. Our findings support the use of FV vectors as a safe and effective alternative to lentiviral vectors for ex vivo transduction of stem cells that are quiescent during culture but divide following transplantation.