bluuebunny/crossref_metadata_2025_split huggingface dataset:
Productive Replication of Adeno-Associated Virus Can Occur in Human Papillomavirus Type 16 (HPV-16) Episome-Containing Keratinocytes and Is Augmented by the HPV-16 E2 Protein
Authors: Ogston Phyllis , Raj Kenneth , Beard Peter 
Published: July 2002

ABSTRACT
We used a sensitive assay to test whether an adeno-associated virus (AAV) productive replication cycle can occur in immortalized human keratinocytes carrying episomal human papillomavirus type 16 (HPV-16) DNA. Following transfection with cloned AAV DNA, infectious AAV was produced, and the infectivity was blocked by anti-AAV antiserum. The HPV-16 E2 protein substantially increased the yield of AAV. Other HPV early proteins did not, in our experiments, show this ability. E2 has been shown to be able to affect p53 levels and to block cell cycle progression at mitosis. We tested the effect of changes in p53 expression on AAV replication and found that large differences in the level of p53 did not alter AAV DNA replication. In extension of this, we found that cellular help for AAV in response to stress was also independent of p53. To test if a mitotic block could trigger AAV DNA replication, we treated the cells with the mitotic inhibitor nocodazole. AAV DNA replication was stimulated by the presence of nocodazole in these and a number of other cell types tested. Yields of infectious virus, however, were not increased by this treatment. We conclude that the HPV-16 E2 protein stimulates AAV multiplication in these cells and propose that this occurs independently of the effects of E2 on p53 and cell cycle progression. Since the effect of E2 was not seen in keratinocytes lacking the HPV-16 episome, we suggest that E2 can help AAV by working in concert with other HPV-16 proteins.
The Role of Alpha/Beta and Gamma Interferons in Development of Immunity to Influenza A Virus in Mice
Authors: Price Graeme E. , Gaszewska-Mastarlarz Anna , Moskophidis Demetrius 
Published: July 2002

ABSTRACTDuring influenza virus infection innate and adaptive immune defenses are activated to eliminate the virus and thereby bring about recovery from illness. Both arms of the adaptive immune system, antibody neutralization of free virus and termination of intracellular virus replication by antiviral cytotoxic T cells (CTLs), play pivotal roles in virus elimination and protection from disease. Innate cytokine responses, such as alpha/beta interferon (IFN-α/β) or IFN-γ, can have roles in determining the rate of virus replication in the initial stages of infection and in shaping the initial inflammatory and downstream adaptive immune responses. The effect of these cytokines on the replication of pneumotropic influenza A virus in the respiratory tract and in the regulation of adaptive antiviral immunity was examined after intranasal infection of mice with null mutations in receptors for IFN-α/β, IFN-γ, and both IFNs. Virus titers in the lungs of mice unable to respond to IFNs were not significantly different from congenic controls for both primary and secondary infection. Likewise the mice were comparably susceptible to X31 (H3N2) influenza virus infection. No significant disruption to the development of normal antiviral CTL or antibody responses was observed. In contrast, mice bearing the disrupted IFN-α/β receptor exhibited accelerated kinetics and significantly higher levels of neutralizing antibody activity during primary or secondary heterosubtypic influenza virus infection. Thus, these observations reveal no significant contribution for IFN-controlled pathways in shaping acute or memory T-cell responses to pneumotropic influenza virus infection but do indicate some role for IFN-α/β in the regulation of antibody responses. Recognizing the pivotal role of CTLs and antibody in virus clearance, it is reasonable to assume a redundancy in IFN-mediated antiviral effects in pulmonary influenza. However, IFN-α/β seems to be a valid factor in determining tissue tropism and replicative rates of highly virulent influenza virus strains as reported previously by others, and this aspect is discussed here.
A Single Intramuscular Injection of Recombinant Plasmid DNA Induces Protective Immunity and Prevents Japanese Encephalitis in Mice
Authors: Chang Gwong-Jen J. , Hunt Ann R. , Davis Brent 
Published: July 2002

ABSTRACTPlasmid vectors containing Japanese encephalitis virus (JEV) premembrane (prM) and envelope (E) genes were constructed that expressed prM and E proteins under the control of a cytomegalovirus immediate-early gene promoter. COS-1 cells transformed with this plasmid vector (JE-4B clone) secreted JEV-specific extracellular particles (EPs) into the culture media. Groups of outbred ICR mice were given one or two doses of recombinant plasmid DNA or two doses of the commercial vaccine JEVAX. All mice that received one or two doses of DNA vaccine maintained JEV-specific antibodies 18 months after initial immunization. JEVAX induced 100% seroconversion in 3-week-old mice; however, none of the 3-day-old mice had enzyme-linked immunosorbent assay titers higher than 1:400. Female mice immunized with this DNA vaccine developed plaque reduction neutralization antibody titers of between 1:20 and 1:160 and provided 45 to 100% passive protection to their progeny following intraperitoneal challenge with 5,000 PFU of virulent JEV strain SA14. Seven-week-old adult mice that had received a single dose of JEV DNA vaccine when 3 days of age were completely protected from a 50,000-PFU JEV intraperitoneal challenge. These results demonstrate that a recombinant plasmid DNA which produced JEV EPs in vitro is an effective vaccine.
Stable High-Level Expression of Heterologous Genes In Vitro and In Vivo by Noncytopathic DNA-Based Kunjin Virus Replicon Vectors
Authors: Varnavski Andrei N. , Young Paul R. , Khromykh Alexander A. 
Published: July 2002

ABSTRACT
Primary features of the flavivirus Kunjin (KUN) subgenomic replicons include continuous noncytopathic replication in host cell cytoplasm and the ability to be encapsidated into secreted virus-like particles (VLPs). Previously we reported preparation of RNA-based KUN replicon vectors and expression of heterologous genes (HG) in cell culture after RNA transfection or after infection with recombinant KUN VLPs (A. N. Varnavski and A. A. Khromykh, Virology 255:366–375, 1999). In this study we describe the development of the next generation of KUN replicon vectors, which allow synthesis of replicon RNA in vivo from corresponding plasmid DNAs. These DNA-based vectors were able to direct stable expression of β-galactosidase (β-Gal) in several mammalian cell lines, and expression remained high (∼150 pg per cell) throughout cell passaging. The applicability of these vectors in vivo was demonstrated by β-Gal expression in the mouse lung epithelium for at least 8 weeks after intranasal inoculation and induction of anti-β-Gal antibody response after intramuscular inoculation of the β-Gal-encoding KUN replicon DNA. The noncytopathic nature of DNA-based KUN replicon vectors combined with high-level and stability of HG expression in a broad range of host cells should prove them to be useful in a variety of applications in vitro and in vivo.
Direct Ex Vivo Measurement of CD8+T-Lymphocyte Responses to Human Parvovirus B19
Authors: Tolfvenstam T. , Oxenius A. , Price D. A. , Shacklett B. L. , Spiegel H. M. L. , Hedman K. , Norbeck O. , Levi M. , Olsen K. , Kantzanou M. , Nixon D. F. , Broliden K. , Klenerman P. 
Published: July 2002

ABSTRACTParvovirus B19 is a common human pathogen which can cause severe syndromes, including aplastic anemia and fetal hydrops. The mapping of the first parvovirus B19-derived CD8+T-lymphocyte epitope is described. This HLA-B35-restricted peptide derives from the nonstructural (NS1) protein and is strongly immunogenic in B19 virus-seropositive donors.
Tracking the Spread of a
lacZ
-Tagged Herpes Simplex Virus Type 1 between the Eye and the Nervous System of the Mouse: Comparison of Primary and Recurrent Infection
Authors: Shimeld Carolyn , Efstathiou Stacey , Hill Terry 
Published: July 2002

ABSTRACT

The spread of herpes simplex virus type 1 (HSV-1) during primary ocular infection and after reactivation of latent infection in the trigeminal ganglion (TG) was examined in the mouse using a genetically modified virus containing the
lacZ
reporter gene under the control of the immediate-early 110 promoter. Whole tissue mounts of the eye and lids, their sensory nerves, and TG with the attached dorsal root entry zone (DRE) into the central nervous system (CNS) were stained for β-galactosidase. Sixteen hours after inoculation of the cornea by scarification, staining was found in the scarified epithelium of the cornea and in the unscarified conjunctiva. By 24 h, staining was also seen in a few TG neurons and by 96 h their number had greatly increased and their distribution was more widespread. Stained cells (identified as Schwann cells by their staining for glial fibrillary acidic protein [GFAP] or S-100) in the TG were first seen close to stained neurons at 40 h, and by 48 h lines of such cells extended partway toward the periphery and toward the DRE. By 72 h, these lines had reached the periphery and the DRE where the adjacent CNS was also stained. In the cornea, stained cells with the morphology and arrangement of Schwann cells were seen from 40 to 120 h. After reactivation of latent infection, 10 of 22 samples had positively stained neurons. In eight samples, corneal and lid epithelial cells were stained. No stained Schwann cells were seen in the TG; however, branched networks of such cells were present in the cornea and the lids. This detailed sequential analysis has provided new information on the involvement of Schwann cells in the pathogenesis of primary and recurrent HSV-1 disease in the TG and the cornea.
Hepatitis B Virus Large Envelope Protein Interacts with γ2-Adaptin, a Clathrin Adaptor-Related Protein
Authors: Hartmann-Stühler Cora , Prange Reinhild 
Published: July 2002

ABSTRACT

For the outcome of a hepatitis B virus (HBV) infection, the viral L envelope protein with its pre-S domain performs pivotal functions by mediating attachment of HBV to liver cells, envelopment of viral capsids, release of (sub)viral particles, regulation of supercoiled DNA amplification, and transcriptional transactivation. To assess its multiple functions and host-protein assistance involved, we initiated a two-hybrid screen using the L-specific pre-S1 domain as bait. With this approach, we have identified γ2-adaptin, a putative member of the clathrin adaptor proteins responsible for protein sorting and trafficking, as a specific binding partner of L protein. Evidence for a physical interaction between L protein and γ2-adaptin was also demonstrated by affinity chromatography and coimmunoprecipitation, and the binding sites were mapped to the L-specific pre-S1 domain and the γ2-adaptin-specific ear domain. The specificity of the interaction was further sustained by the failure of γ1-adaptin, a closely related γ2-adaptin homologue, to associate with L protein. Analysis of an L mutant protein indicates that the L–γ2-adaptin interaction strictly depends on the pre-S1 domain of transmembrane L protein oriented to the cytosol and thus appears to occur in the cytosolic environment. Interestingly, coexpression of the two interacting partners in transfected cells resulted in recruitment of γ2-adaptin by L protein onto
cis
-Golgi-like structures, strongly indicating that the association is physiologically relevant. Together, the results suggest a role for γ2-adaptin in L-mediated processes of viral biogenesis and/or pathogenesis, such as facilitating and guiding HBV assembly.
Physical and Functional Interactions between the Corepressor CtBP and the Epstein-Barr Virus Nuclear Antigen EBNA3C
Authors: Touitou Robert , Hickabottom Mark , Parker Gillian , Crook Tim , Allday Martin J. 
Published: July 2002

ABSTRACT

CtBP has been shown to be a highly conserved corepressor of transcription. E1A and all the various transcription factors to which CtBP binds contain a conserved PLDLS CtBP-interacting domain, and EBNA3C includes a PLDLS motif (amino acids [aa] 728 to 732). Here we show that EBNA3C binds to CtBP both in vitro and in vivo and that the interaction requires an intact PLDLS. The C terminus of EBNA3C (aa 580 to 992) has modest
trans
-repressor activity when it is fused to the DNA-binding domain of Gal4, and deletion or mutation of the PLDLS sequence ablates this and unmasks a transactivation function within the fragment. However, loss of the CtBP interaction motif had little effect on the ability of full-length EBNA3C to repress transcription. A striking correlation between CtBP binding and the capacity of EBNA3C to cooperate with (Ha-)Ras in the immortalization and transformation of primary rat embryo fibroblasts was also revealed.
Specific Interaction of a Novel Foamy Virus Env Leader Protein with the N-Terminal Gag Domain
Authors: Wilk Thomas , Geiselhart Verena , Frech Matthias , Fuller Stephen D. , Flügel Rolf M. , Löchelt Martin 
Published: July 2002

ABSTRACT
Cryoelectron micrographs of purified human foamy virus (HFV) and feline foamy virus (FFV) particles revealed distinct radial arrangements of Gag proteins. The capsids were surrounded by an internal Gag layer that in turn was surrounded by, and separated from, the viral membrane. The width of this layer was about 8 nm for HFV and 3.8 nm for FFV. This difference in width is assumed to reflect the different sizes of the HFV and FFV MA domains: the HFV MA domain is about 130 residues longer than that of FFV. The distances between the MA layer and the edge of the capsid were identical in different particle classes. In contrast, only particles with a distended envelope displayed an invariant, close spacing between the MA layer and the Env membrane which was absent in the majority of particles. This indicates a specific interaction between MA and Env at an unknown step of morphogenesis. This observation was supported by surface plasmon resonance studies. The purified N-terminal domain of FFV Gag specifically interacted with synthetic peptides and a defined protein domain derived from the N-terminal Env leader protein. The specificity of this interaction was demonstrated by using peptides varying in the conserved Trp residues that are known to be required for HFV budding. The interaction with Gag required residues within the novel virion-associated FFV Env leader protein of about 16.5 kDa.
Rescue of Recombinant
Thogoto Virus
from Cloned cDNA
Authors: Wagner Elke , Engelhardt Othmar G. , Gruber Simone , Haller Otto , Kochs Georg 
Published: July 2002

ABSTRACT

Thogoto virus
(THOV) is a tick-transmitted orthomyxovirus with a genome consisting of six negative-stranded RNA segments. To rescue a recombinant THOV, the viral structural proteins were produced from expression plasmids by means of a vaccinia virus expressing the T7 RNA polymerase. Genomic virus RNAs (vRNAs) were generated from plasmids under the control of the RNA polymerase I promoter. Using this system, we could efficiently recover recombinant THOV following transfection of 12 plasmids into 293T cells. To verify the recombinant nature of the rescued virus, specific genetic tags were introduced into two vRNA segments. The availability of this efficient reverse genetics system will allow us to address hitherto-unanswered questions regarding the biology of THOV by manipulating viral genes in the context of infectious virus.
Localization to the Nucleolus Is a Common Feature of Coronavirus Nucleoproteins, and the Protein May Disrupt Host Cell Division
Authors: Wurm Torsten , Chen Hongying , Hodgson Teri , Britton Paul , Brooks Gavin , Hiscox Julian A. 
Published: July 2002

ABSTRACT

The subcellular localization of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) (group I and group II coronaviruses, respectively) nucleoproteins (N proteins) were examined by confocal microscopy. The proteins were shown to localize either to the cytoplasm alone or to the cytoplasm and a structure in the nucleus. This feature was confirmed to be the nucleolus by using specific antibodies to nucleolin, a major component of the nucleolus, and by confocal microscopy to image sections through a cell expressing N protein. These findings are consistent with our previous report for infectious bronchitis virus (group III coronavirus) (J. A. Hiscox et al.
,
J. Virol. 75:506–512, 2001), indicating that nucleolar localization of the N protein is a common feature of the coronavirus family and is possibly of functional significance. Nucleolar localization signals were identified in the domain III region of the N protein from all three coronavirus groups, and this suggested that transport of N protein to the nucleus might be an active process. In addition, our results suggest that the N protein might function to disrupt cell division. Thus, we observed that approximately 30% of cells transfected with the N protein appeared to be undergoing cell division. The most likely explanation for this is that the N protein induced a cell cycle delay or arrest, most likely in the G
2
/M phase. In a fraction of transfected cells expressing coronavirus N proteins, we observed multinucleate cells and dividing cells with nucleoli (which are only present during interphase). These findings are consistent with the possible inhibition of cytokinesis in these cells.
Identification of a Central DNA Flap in Feline Immunodeficiency Virus
Authors: Whitwam Todd , Peretz Mary , Poeschla Eric 
Published: July 2002

ABSTRACT

A duplication of the polypurine tract (PPT) at the center of the human immunodeficiency virus type 1 (HIV-1) genome (the cPPT) has been shown to prime a separate plus-strand initiation and to result in a plus-strand displacement (DNA flap) that plays a role in nuclear import of the viral preintegration complex. Feline immunodeficiency virus (FIV) is a lentivirus that infects nondividing cells, causes progressive CD4
+
T-cell depletion, and has been used as a substrate for lentiviral vectors. However, the PPT sequence is not duplicated elsewhere in the FIV genome and a central plus-strand initiation or strand displacement has not been identified. Using Southern blotting of S1 nuclease-digested FIV preintegration complexes isolated from infected cells, we detected a single-strand discontinuity at the approximate center of the reverse-transcribed genome. Primer extension analyses assigned the gap to the plus strand, and mapped the 5′ terminus of the downstream (D+) segment to a guanine residue in a purine-rich tract in
pol
(AAAAGAAGAGGTA
G
GA). RACE experiments then mapped the 3′ terminus of the upstream plus (U+)-strand segment to a T nucleotide located 88 nucleotides downstream of the D+ strand 5′ terminus, thereby identifying the extent of D+ strand displacement and the central termination sequence of this virus. Unlike HIV, the FIV cPPT is significantly divergent in sequence from its 3′ counterpart (AAAAAAGAAAAAAGGGTGG) and contains one and in some cases two pyrimidines. An invariant thymidine located −2 to the D+ strand origin is neither required nor optimal for codon usage at this position. Although the mapped cPPTs of FIV and HIV-1 act in
cis
, they encode homologous amino acids in integrase.
Arginine-Glycine-Aspartic Acid Motif Is Critical for Human Parechovirus 1 Entry
Authors: Boonyakiat Yingmanee , Hughes Pamela J. , Ghazi Farideh , Stanway Glyn 
Published: July 2002

ABSTRACT
The human parechovirus 1 RGD motif in VP1 was studied by mutagenesis. An RGD-to-RGE change gave only revertant viruses with a restored RGD, while deletion of GD was lethal and nonrevertable. Mutations at the +1 and +2 positions had some effect on growth properties and a +1 M-to-P change was lethal. These studies indicate that the RGD motif plays a critical role in infectivity, presumably by interacting with integrins, and that downstream amino acids can have an influence on function.
Resistance to Nucleoside Analog Reverse Transcriptase Inhibitors Mediated by Human Immunodeficiency Virus Type 1 p6 Protein
Authors: Peters Solange , Muñoz Miguel , Yerly Sabine , Sanchez-Merino Victor , Lopez-Galindez Cecilio , Perrin Luc , Larder Brendan , Cmarko Dusan , Fakan Stanislav , Meylan Pascal , Telenti Amalio 
Published: July 2002

ABSTRACT

Resistance of human immunodeficiency virus type 1 (HIV-1) to antiretroviral agents results from target gene mutation within the
pol
gene, which encodes the viral protease, reverse transcriptase (RT), and integrase. We speculated that mutations in genes other that the drug target could lead to drug resistance. For this purpose, the p1-p6

gag

-p6

pol

region of HIV-1, placed immediately upstream of
pol
, was analyzed. This region has the potential to alter Pol through frameshift regulation (
p1
), through improved packaging of viral enzymes (p6
Gag
), or by changes in activation of the viral protease (p6
Pol
). Duplication of the proline-rich p6
Gag
PTAP motif, necessary for late viral cycle activities, was identified in plasma virus from 47 of 222 (21.2%) patients treated with nucleoside analog RT inhibitor (NRTI) antiretroviral therapy but was identified very rarely from drug-naı̈ve individuals. Molecular clones carrying a 3-amino-acid duplication, APPAPP (transframe duplication SPTSPT in p6
Pol
), displayed a delay in protein maturation; however, they packaged a 34% excess of RT and exhibited a marked competitive growth advantage in the presence of NRTIs. This phenotype is reminiscent of the inoculum effect described in bacteriology, where a larger input, or a greater infectivity of an organism with a wild-type antimicrobial target, leads to escape from drug pressure and a higher MIC in vitro. Though the mechanism by which the PTAP region participates in viral maturation is not known, duplication of this proline-rich motif could improve assembly and packaging at membrane locations, resulting in the observed phenotype of increased infectivity and drug resistance.
Interleukin-7 in Plasma Correlates with CD4 T-Cell Depletion and May Be Associated with Emergence of Syncytium-Inducing Variants in Human Immunodeficiency Virus Type 1-Positive Individuals
Authors: Llano Anuska , Barretina Jordi , Gutiérrez Arantxa , Blanco Julià , Cabrera Cecilia , Clotet Bonaventura , Esté José A. 
Published: July 2002

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) primary infection is characterized by the use of CCR5 as a coreceptor for viral entry, which is associated with the non-syncytium-inducing (NSI) phenotype in lymphoid cells. Syncytium-inducing (SI) variants of HIV-1 appear in advanced stages of HIV-1 infection and are characterized by the use of CXCR4 as a coreceptor. The emergence of SI variants is accompanied by a rapid decrease in the number of T cells. However, it is unclear why SI variants emerge and what factors trigger the evolution of HIV from R5 to X4 variants. Interleukin-7 (IL-7), a cytokine produced by stromal cells of the thymus and bone marrow and by keratin, is known to play a key role in T-cell development. We evaluated IL-7 levels in plasma of healthy donors and HIV-positive patients and found significantly higher levels in HIV-positive patients. There was a negative correlation between circulating IL-7 levels and CD4+T-cell count in HIV-positive patients (r= −0.621;P< 0.001), suggesting that IL-7 may be involved in HIV-induced T-cell depletion and disease progression. IL-7 levels were higher in individuals who harbored SI variants and who had progressed to having CD4 cell counts of lower than 200 cells/μl than in individuals with NSI variants at a similar stage of disease. IL-7 induced T-cell proliferation and up-regulated CXCR4 expression in peripheral blood mononuclear cells in vitro. Taken together, our results suggest a role for IL-7 in the maintenance of T-cell regeneration and depletion by HIV in infected individuals and a possible relationship between IL-7 levels and the emergence of SI variants.