bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA-Functionalized Nanotube Membranes with Single-Base Mismatch Selectivity
Authors: Kohli Punit , Harrell C. Chad , Cao Zehui , Gasparac Rahela , Tan Weihong , Martin Charles R.
Published: August 2004

We describe synthetic membranes in which the molecular recognition chemistry used to accomplish selective permeation is DNA hybridization. These membranes contain template-synthesized gold nanotubes with inside diameters of 12 nanometers, and a “transporter” DNA-hairpin molecule is attached to the inside walls of these nanotubes. These DNA-functionalized nanotube membranes selectively recognize and transport the DNA strand that is complementary to the transporter strand, relative to DNA strands that are not complementary to the transporter. Under optimal conditions, single-base mismatch transport selectivity can be obtained.
Simulations of Jets Driven by Black Hole Rotation
Authors: Semenov Vladimir , Dyadechkin Sergey , Punsly Brian
Published: August 2004

The origin of jets emitted from black holes is not well understood; however, there are two possible energy sources: the accretion disk or the rotating black hole. Magnetohydrodynamic simulations show a well-defined jet that extracts energy from a black hole. If plasma near the black hole is threaded by large-scale magnetic flux, it will rotate with respect to asymptotic infinity, creating large magnetic stresses. These stresses are released as a relativistic jet at the expense of black hole rotational energy. The physics of the jet initiation in the simulations is described by the theory of black hole gravitohydromagnetics.
Our Once and Future Fate
Authors: Kinzig Ann
Published: August 2004

One with Nineveh
Politics, Consumption, and the Human Future. by Paul R. Ehrlich and Anne H. Ehrlich. Island Press, Washington, DC, 2004. 459 pp. $27. ISBN 1-55963-879-6.

Paying particular attention to three crucial factors--increasing human population, rising consumption, and accelerating political and economic inequity--that threaten Earth's ability to sustain us, the authors explore some avenues that hold hopes for reversing current trends.
Two-Step Synthesis of Carbohydrates by Selective Aldol Reactions
Authors: Northrup Alan B. , MacMillan David W. C.
Published: August 2004

Studies of carbohydrates have been hampered by the lack of chemical strategies for the expeditious construction and coupling of differentially protected monosaccharides. Here, a synthetic route based on aldol coupling of three aldehydes is presented for the de novo production of polyol differentiated hexoses in only two chemical steps. The dimerization of α-oxyaldehydes, catalyzed by
l
-proline, is then followed by a tandem Mukaiyama aldol addition-cyclization step catalyzed by a Lewis acid. Differentially protected glucose, allose, and mannose stereoisomers can each be selected, in high yield and stereochemical purity, simply by changing the solvent and Lewis acid used. The reaction sequence also efficiently produces
13
C-labeled analogs, as well as structural variants such as 2-amino– and 2-thio–substituted derivatives.
Substructure in the Circumstellar Disk Around the Young Star AU Microscopii
Authors: Liu Michael C.
Published: August 2004

Keck adaptive optics imaging with a physical resolution of 0.4 astronomical units (AU) resolves the inner (15 to 80 AU) disk of AU Microscopii (AU Mic, GJ 803, HD 197481), the nearest known scattered light disk to Earth. The inner disk is asymmetric and possesses a sharp change in structure at 35 AU. The disk also shows spatially localized enhancements and deficits at 25- to 40-AU separations. The overall morphology points to the influence of unseen larger bodies and resembles structures expected from recent planet formation. AU Mic is coeval with the archetypical debris disk system β Pictoris, and the similarities between their two disks point to synchronous disk evolution. Multiple indications of substructure appear to be common in circumstellar disks at an age of ≈12 million years.
Sustainable Hydrogen Production
Authors: Turner John A.
Published: August 2004

Identifying and building a sustainable energy system are perhaps two of the most critical issues that today's society must address. Replacing our current energy carrier mix with a sustainable fuel is one of the key pieces in that system. Hydrogen as an energy carrier, primarily derived from water, can address issues of sustainability, environmental emissions, and energy security. Issues relating to hydrogen production pathways are addressed here. Future energy systems require money and energy to build. Given that the United States has a finite supply of both, hard decisions must be made about the path forward, and this path must be followed with a sustained and focused effort.
Replication and Cytopathic Effect of Oncolytic Vesicular Stomatitis Virus in Hypoxic Tumor Cells In Vitro and In Vivo
Authors: Connor John H. , Naczki Christine , Koumenis Costas , Lyles Douglas S.
Published: August 2004

Tumor hypoxia presents an obstacle to the effectiveness of most antitumor therapies, including treatment with oncolytic viruses. In particular, an oncolytic virus must be resistant to the inhibition of DNA, RNA, and protein synthesis that occurs during hypoxic stress. Here we show that vesicular stomatitis virus (VSV), an oncolytic RNA virus, is capable of replication under hypoxic conditions. In cells undergoing hypoxic stress, VSV infection produced larger amounts of mRNA than under normoxic conditions. However, translation of these mRNAs was reduced at earlier times postinfection in hypoxia-adapted cells than in normoxic cells. At later times postinfection, VSV overcame a hypoxia-associated increase in α subunit of eukaryotic initiation factor 2 (eIF-2α) phosphorylation and initial suppression of viral protein synthesis in hypoxic cells to produce large amounts of viral protein. VSV infection caused the dephosphorylation of the translation initiation factor eIF-4E and inhibited host translation similarly under both normoxic and hypoxic conditions. VSV produced progeny virus to similar levels in hypoxic and normoxic cells and showed the ability to expand from an initial infection of 1% of hypoxic cells to spread through an entire population. In all cases, virus infection induced classical cytopathic effects and apoptotic cell death. When VSV was used to treat tumors established in nude mice, we found VSV replication in hypoxic areas of these tumors. This occurred whether the virus was administered intratumorally or intravenously. These results show for the first time that VSV has an inherent capacity for infecting and killing hypoxic cancer cells. This ability could represent a critical advantage over existing therapies in treating established tumors.
Characterization of a Highly Pathogenic Molecular Clone of Feline Immunodeficiency Virus Clade C
Authors: de Rozières Sohela , Mathiason Candace K. , Rolston Matthew R. , Chatterji Udayan , Hoover Edward A. , Elder John H.
Published: August 2004

We have derived and characterized a highly pathogenic molecular isolate of feline immunodeficiency virus subtype C (FIV-C) CABCpady00C. Clone FIV-C36 was obtained by lambda cloning from cats that developed severe immunodeficiency disease when infected with CABCpady00C (Abbotsford, British Columbia, Canada). Clone FIV-C36 Env is 96% identical to the noninfectious FIV-C isolate sequence deposited in GenBank (FIV-Cgb; GenBank accession number
AF474246
) (A. Harmache et al.) but is much more divergent in Env when compared to the subgroup A clones Petaluma (34TF10) and FIV-PPR (76 and 78% divergence, respectively). Clone FIV-C36 was able to infect freshly isolated feline peripheral blood mononuclear cells and primary T-cell lines but failed to productively infect CrFK cells, as is typical of FIV field isolates. Two-week-old specific-pathogen-free cats infected with FIV-C36 tissue culture supernatant became PCR positive and developed severe acute immunodeficiency disease similar to that caused by the uncloned CABCpady00C parent. At 4 to 5 weeks postinfection (PI), 3 of 4 animals developed CD4
+
-T-cell depletion, fever, weight loss, diarrhea, and opportunistic infections, including ulcerative stomatitis and tonsillitis associated with abundant bacterial growth, pneumonia, and pyelonephritis, requiring euthanasia. Histopathology confirmed severe thymic and systemic lymphoid depletion. Interestingly, the dam also became infected with a high viral load at 5 weeks PI of the kittens and developed a similar disease syndrome, requiring euthanasia at 11 weeks PI of the kittens. This constitutes the first report of a replication-competent, infectious, and pathogenic molecular clone of FIV-C. Clone FIV-C36 will facilitate dissection of the pathogenic determinants of FIV.
Measles Virus (MV) Hemagglutinin: Evidence that Attachment Sites for MV Receptors SLAM and CD46 Overlap on the Globular Head
Authors: Massé Nicolas , Ainouze Michelle , Néel Benjamin , Wild T. Fabian , Buckland Robin , Langedijk Johannes P. M.
Published: August 2004

Measles virus hemagglutinin (MVH) residues potentially responsible for attachment to the wild-type (wt) MV receptor SLAM (CD150) have been identified and localized on the MVH globular head by reference to a revised hypothetical structural model for MVH (
www.pepscan.nl/downloads/measlesH.pdb
). We show that the mutation of five charged MVH residues which are conserved among morbillivirus H proteins has major effects on both SLAM downregulation and SLAM-dependent fusion. In the three-dimensional surface representation of the structural model, three of these residues (D505, D507, and R533) align the rim on one side of the cavity on the top surface of the MVH globular head and form the basis of a single continuous site that overlaps with the 546-548-549 CD46 binding site. We show that the overlapping sites fall within the footprint of an anti-MVH monoclonal antibody that neutralizes both wt and laboratory-vaccine MV strains and whose epitope contains R533. Our study does not exclude the possibility that Y481 binds CD46 directly but suggests that the N481Y mutation of wt MVH could influence, at a distance, the conformation of the overlapping sites so that affinity to CD46 increases. The relevance of these results to present concepts of MV receptor usage is discussed, and an explanation is proposed as to why morbillivirus attachment proteins are H, whereas those from the other paramyxoviruses are HN (hemagglutinin-neuraminidase).
trans
-Complementation Allows Recovery of Human Respiratory Syncytial Viruses That Are Infectious but Deficient in Cell-to-Cell Transmission
Authors: Oomens A. G. P. , Wertz Gail W.
Published: August 2004

Human respiratory syncytial virus (HRSV) expresses three transmembrane glycoproteins: small hydrophobic protein SH, attachment protein G, and fusion protein F. The genes encoding SH and G can be deleted from the HRSV genome and infectious virus recovered. In contrast, HRSVs lacking the F gene or a functional replacement thereof have not been reported. To generate a system with which to study the roles of the viral transmembrane glycoproteins, including F, in the HRSV life cycle, we generated a cell line expressing a heterologous viral glycoprotein for complementation of glycoprotein function in
trans
. We previously demonstrated that the baculovirus GP64 protein or a chimeric form of GP64 carrying the 12 C-terminal amino acids of the HRSV F protein (GP
64/F
) can efficiently mediate HRSV infectivity and improve its stability, when expressed from an engineered HRSV genome. Here, we report the development of a stably transfected Vero cell line (Vbac) constitutively expressing the GP
64/F
protein. From the Vbac cell line, viruses that lacked the SH and F open reading frames (ORFs) or the SH, G, and F ORFs could be recovered from cDNAs. These viruses, designated RSΔ
sh,f
and RSΔ
sh,g,f
, respectively, had place-keeper ORFs inserted in place of the deleted ORFs to maintain authentic transcription levels. In the Vbac cell line, RSΔ
sh,f
and RSΔ
sh,g,f
could be amplified to near wild-type-level titers, and the resulting viruses were infectious to Vero and HEp-2 cells. After entry into Vero or HEp-2 cells, however, neither virus RSΔ
sh,f
nor virus RSΔ
sh,g,f
was able to spread in the infected cultures. Growth analyses showed that infectious virions were not produced in Vero or HEp-2 cells infected with RSΔ
sh,f
and RSΔ
sh,g,f
. Combined, these data provide direct evidence that HRSV F is an essential viral protein required for cell-to-cell transmission and demonstrate that complementation with the GP64 protein in
trans
constitutes a powerful tool for the study of the role of individual HRSV transmembrane glycoproteins in virus assembly, morphogenesis, and pathogenesis. In addition, the ability to generate infectious but nonspreading viruses may provide an alternative approach for the development of safe and stable HRSV vaccine candidates.
Adjuvant Activities of Novel Cytokines, Interleukin-23 (IL-23) and IL-27, for Induction of Hepatitis C Virus-Specific Cytotoxic T Lymphocytes in HLA-A*0201 Transgenic Mice
Authors: Matsui Masanori , Moriya Osamu , Belladonna Maria Laura , Kamiya Sadahiro , Lemonnier François A. , Yoshimoto Takayuki , Akatsuka Toshitaka
Published: August 2004

Searching the sequence databases has revealed two novel cytokines: interleukin-23 (IL-23) and IL-27. These cytokines are quite similar to, but clearly distinct from IL-12 in their structures and T-cell stimulatory fashions. In contrast to IL-12, however, little is known about the roles of IL-23 and IL-27 in the immune regulation. Previously, we evaluated the prime-boost immunization consisting of priming and the first boosting with the hepatitis C virus (HCV)-core expression plasmid, followed by a second boosting with recombinant adenovirus expressing HCV core for induction of HCV core-specific cytotoxic T lymphocytes (CTLs) in BALB/c mice. The present study demonstrates that HCV-specific CTL induction was greatly enhanced by coinoculation of an IL-12 expression plasmid in the prime-boost immunization, indicating the potent adjuvant activity of IL-12. We investigated whether similar adjuvant effects could be exerted by either IL-23 or IL-27 in a prime-boost immunization with HLA-A*0201 transgenic mice. Coadministration of either an IL-23 or an IL-27 expression plasmid, as well as an IL-12 expression plasmid, in a prime-boost immunization enhanced induction of HCV-specific CTLs and led to dramatic increases in the numbers of gamma interferon (IFN-γ)-producing, HCV-specific CD8+cells. Further, preinjections of IL-12, IL-23, or IL-27 expression plasmids before immunization resulted in great increases in the number of IFN-γ-producing, HCV-specific CD8+cells in response to immunization with recombinant adenovirus. These data revealed that both IL-23 and IL-27, as well as IL-12, are potent adjuvants for epitope-specific CTL induction. The two novel cytokines might offer new prophylactic and therapeutic strategies against infectious pathogens such as HCV.
Analysis of Human Immunodeficiency Virus Type 1 Transcriptional Elongation in Resting CD4+T Cells In Vivo
Authors: Lassen Kara G. , Bailey Justin R. , Siliciano Robert F.
Published: August 2004

A stable latent reservoir for human immunodeficiency virus type 1 (HIV-1) in resting memory CD4+T cells presents a barrier to eradication of the infection even in patients on highly active antiretroviral therapy. Potential mechanisms for latency include inaccessibility of the integrated viral genome, absence of key host transcription factors, premature termination of HIV-1 RNAs, and abnormal splicing patterns. To differentiate among these mechanisms, we isolated extremely pure populations of resting CD4+T cells from patients on highly active antiretroviral therapy. These cells did not produce virus but retained the capacity to do so if appropriately stimulated. Products of HIV-1 transcription were examined in purified resting CD4+T cells. Although short, prematurely terminated HIV-1 transcripts have been suggested as a marker for latently infected cells, the production of short transcripts had not been previously demonstrated in purified populations of resting CD4+T cells. By separating RNA into polyadenylated and nonpolyadenylated fractions, we showed that resting CD4+T cells from patients on highly active antiretroviral therapy produce abortive transcripts that lack a poly(A) tail and that terminate prior to nucleotide 181. Short transcripts dominated the pool of total HIV-1 transcripts in resting CD4+T cells. Processive, polyadenylated HIV-1 mRNAs were also present at a low level. Both unspliced and multiply spliced forms were found. Taken together, these results show that the nonproductive nature of the infection in resting CD4+T cells from patients on highly active antiretroviral therapy is not due to absolute blocks at the level of either transcriptional initiation or elongation but rather relative inefficiencies at multiple steps.
Conformation- and Fusion-Defective Mutations in the Hypothetical Phospholipid-Binding and Fusion Peptides of Viral Hemorrhagic Septicemia Salmonid Rhabdovirus Protein G
Authors: Rocha A. , Ruiz S. , Tafalla C. , Coll J. M.
Published: August 2004

Fourteen single and two double point mutants in the highly conserved region (positions 56 to 159) of the G gene of viral hemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were selected and obtained in plasmids by site-directed mutagenesis. Fish cell monolayers transfected with the mutant plasmids were then assayed for protein G (pG) expression, conformation-dependent monoclonal antibody (MAb) reactivity, and cell-cell fusion. Some mutations located in the phospholipid-binding p2 peptide (positions 82 to 110; mutants P86A, A96E, G98A, and R107A) abolished both MAb recognition and fusion activity, while others (P79A, L85S, and R103A) abolished MAb recognition but retained fusion at similar or lower pHs compared to those for the wild type. Phospholipid-binding assays of p2-derived synthetic peptides suggested that phosphatidylserine binding was not affected by the mutations studied. On the other hand, three (P79A, L85S, and T135E) of the four mutants retaining fusion activity mapped around two locations showing amino acid variation in 22 VHSV isolates and in neutralizing MAb-resistant mutants described previously. Mutations located in the hypothetical fusion peptide (positions 142 to 159; mutants F147K, P148K, and W154K) abolished both MAb recognition and fusion activity. The existence of mutants with altered conformation and defective fusion in both p2 and fusion peptides provides further evidence in favor of the participation of these and adjacent regions in some of the steps of the VHSV fusion processes, as suggested by previous studies. In addition, because the studied region induced strong immunological responses in trout, some of the mutants described here might be used to design attenuated VHSV vaccines.
Identification of
cis
Sequences Required for Lytic DNA Replication and Packaging of Murine Gammaherpesvirus 68
Authors: Deng Hongyu , Chu Julia T. , Park No-Hee , Sun Ren
Published: August 2004

Human gammaherpesviruses are associated with lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) infection of mice has emerged as a model for understanding gammaherpesvirus pathogenesis in vivo. In contrast to human gammaherpesviruses, MHV-68 replicates in permissive cell lines in a robust manner, presenting an efficient model to study the basic mechanisms for DNA replication and recombination processes. In addition, MHV-68 also infects a broad range of cells of different tissue types and from different host species, and the viral genome persists as an episome in infected cells. These features make MHV-68 an attractive system on which to build gene delivery vectors. We have therefore undertaken a study to identify the
cis
elements required for MHV-68 genome replication and packaging. Here we report that an 8.4-kb MHV-68 genomic fragment between ORF66 and ORF73 conferred on the plasmid the ability to replicate; replication required the presence of either de novo viral infection or viral reactivation from latency. We further mapped the origin of lytic replication (
ori
Lyt) to a 1.25-kb region. Moreover, we demonstrated that the terminal repeat of the viral genome is sufficient for packaging of the replicated
ori
Lyt plasmid into mature viral particles. Functional identification of the MHV-68
ori
Lyt and packaging signal has laid a foundation for investigating the mechanisms controlling gammaherpesvirus DNA replication during the viral lytic phase and will also serve as a base on which to design gene delivery vectors.
The PA Subunit Is Required for Efficient Nuclear Accumulation of the PB1 Subunit of the Influenza A Virus RNA Polymerase Complex
Authors: Fodor Ervin , Smith Matt
Published: August 2004

The RNA genome of influenza virus is transcribed and replicated by the viral RNA polymerase complex in the cell nucleus. We have generated green fluorescent protein (GFP)-tagged polymerase subunits to study the assembly of the polymerase complex. Our results show that individually expressed polymerase basic protein 1 (PB1) and polymerase acidic protein (PA) subunits were distributed in both the cytoplasm and the nucleus, while the polymerase basic protein 2 (PB2) subunit accumulated in the nucleus. Although it has been reported that PB1 alone accumulates in the nucleus, we demonstrate that PB1 requires the coexpression of PA for efficient nuclear accumulation. Our results support a model which proposes that PB1 and PA are transported into the nucleus as a complex.
R5 Human Immunodeficiency Virus Type 1 (HIV-1) Replicates More Efficiently in Primary CD4+T-Cell Cultures Than X4 HIV-1
Authors: Schweighardt Becky , Roy Ann-Marie , Meiklejohn Duncan A. , Grace Edward J. , Moretto Walter J. , Heymann Jonas J. , Nixon Douglas F.
Published: August 2004

In this report, we present evidence that R5 human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in primary CD4+T cells than X4 HIV-1. By comparing CD3/CD28-costimulated CD4+T-cell cultures infected by several X4 and R5 HIV-1 strains, we determined that R5-infected CD4+T cells produce more virus over time than X4-infected CD4+T cells. In the first comparison, we found that more cells were infected by the X4-tropic strain LAI than by the R5-tropic strain JR-CSF and yet that higher levels of viral production were detected in the R5-infected cultures. The differential viral production was partially due to the severe cytopathic effects of the X4 virus. We also compared cultures infected with the isogenic HIV-1 strains NL4-3 (X4) and 49.5 (R5). We found that fewer cells were infected by the R5 strain, and yet similar levels of viral production were detected in both infected cultures. Cell death played less of a role in the differential viral production of these strains, as the cell viability remained comparable in both X4- and R5-infected cultures over time. The final comparison involved the primary R5-tropic isolate KP1 and the primary dual-tropic isolate KP2. Although both strains infected similar numbers of cells and induced comparable levels of cytopathicity, viral production was considerably higher in the R5-infected culture. In summary, these data demonstrate that R5 HIV-1 has an increased capacity to replicate in costimulated CD4+T cells compared to X4 HIV-1.
Codon Optimization of the Tat Antigen of Human Immunodeficiency Virus Type 1 Generates Strong Immune Responses in Mice following Genetic Immunization
Authors: Ramakrishna Lakshmi , Anand Krishnamurthy Kumar , Mohankumar Kumarasamypet M. , Ranga Udaykumar
Published: August 2004

DNA vaccines have been successful in eliciting potent immune responses in mice. Their efficiency, however, is restricted in larger animals. One reason for the limited performance of the DNA vaccines is the lack of molecular strategies to enhance immune responses. Additionally, genes directly cloned from pathogenic organisms may not be efficiently translated in a heterologous host expression system as a consequence of codon bias. To evaluate the influence of codon optimization on the immune response, we elected to use the Tat antigens of human immunodeficiency virus type 1 (HIV-1) (subtype C) and HIV-2, as these viral antigens are poorly immunogenic in natural infection and in experimental immunization and they are functionally important in viral infectivity and pathogenesis. Substituting codons that are optimally used in the mammalian system, we synthetically assembled Tat genes and compared them with the wild-type counterparts in two different mouse strains. Codon-optimized Tat genes induced qualitatively and quantitatively superior immune responses as measured in a T-cell proliferation assay, enzyme-linked immunospot assay, and chromium release assay. Importantly, while the wild-type genes promoted a mixed Th1-Th2-type cytokine profile, the codon-optimized genes induced a predominantly Th1 profile. Using a pepscan strategy, we mapped an immunodominant T-helper epitope to the core and basic domains of HIV-1 Tat. We also identified cross-clade immune responses between HIV-1 subtype B and C Tat proteins mapped to this T-helper epitope. Developing molecular strategies to optimize the immunogenicity of DNA vaccines is critical for inducing strong immune responses, especially to antigens like Tat. Our identification of a highly conserved T-helper epitope in the first exon of HIV-1 Tat of subtype C and the demonstration of a cross-clade immune response between subtypes B and C are important for a more rational design of an HIV vaccine.
Generation of a Latency-Deficient Gammaherpesvirus That Is Protective against Secondary Infection
Authors: Rickabaugh Tammy M. , Brown Helen J. , Martinez-Guzman DeeAnn , Wu Ting-Ting , Tong Leming , Yu Fuqu , Cole Steven , Sun Ren
Published: August 2004

Kaposi's sarcoma-associated herpesvirus and murine gammaherpesvirus-68 (MHV-68) establish latent infections and are associated with various types of malignancies. They are members of the gamma-2 herpesvirus subfamily and encode a replication and transcriptional activator, RTA, which is necessary and sufficient to disrupt latency and initiate the viral lytic cycle in vitro. We have constructed a recombinant MHV-68 virus that overexpresses RTA. This virus has faster replication kinetics in vitro and in vivo, is deficient in establishing latency, exhibits a reduction in the development of a mononucleosis-like disease in mice, and can protect mice against challenge by wild-type MHV-68. The present study, by using MHV-68 as an in vivo model system, demonstrated that RTA plays a critical role in the control of viral latency and suggests that latency is a determinant of viral pathogenesis in vivo.
Hepatitis C Virus E2 Has Three Immunogenic Domains Containing Conformational Epitopes with Distinct Properties and Biological Functions
Authors: Keck Zhen-Yong , Op De Beeck Anne , Hadlock Kenneth G. , Xia Jinming , Li Ta-Kai , Dubuisson Jean , Foung Steven K. H.
Published: August 2004

Mechanisms of virion attachment, interaction with its receptor, and cell entry are poorly understood for hepatitis C virus (HCV) because of a lack of an efficient and reliable in vitro system for virus propagation. Infectious HCV retroviral pseudotype particles (HCVpp) were recently shown to express native E1E2 glycoproteins, as defined in part by HCV human monoclonal antibodies (HMAbs) to conformational epitopes on E2, and some of these antibodies block HCVpp infection (A. Op De Beeck, C. Voisset, B. Bartosch, Y. Ciczora, L. Cocquerel, Z. Y. Keck, S. Foung, F. L. Cosset, and J. Dubuisson, J. Virol.
78:
2994-3002, 2004). Why some HMAbs are neutralizing and others are nonneutralizing is looked at in this report by a series of studies to determine the expression of their epitopes on E2 associated with HCVpp and the role of antibody binding affinity. Antibody cross-competition defined three E2 immunogenic domains with neutralizing HMAbs restricted to two domains that were also able to block E2 interaction with CD81, a putative receptor for HCV. HCVpp immunoprecipitation showed that neutralizing and nonneutralizing domains are expressed on E2 associated with HCVpp, and affinity studies found moderate-to-high-affinity antibodies in all domains. These findings support the perspective that HCV-specific epitopes are responsible for functional steps in virus infection, with specific antibodies blocking distinct steps of virus attachment and entry, rather than the perspective that virus neutralization correlates with increased antibody binding to any virion surface site, independent of the epitope recognized by the antibody. Segregation of virus neutralization and sensitivity to low pH to specific regions supports a model of HCV E2 immunogenic domains similar to the antigenic structural and functional domains of other flavivirus envelope E glycoproteins.
Characterization of Nonpathogenic, Live, Viral Vaccine Vectors Inducing Potent Cellular Immune Responses
Authors: Publicover Jean , Ramsburg Elizabeth , Rose John K.
Published: August 2004

Experimental vaccines based on recombinant vesicular stomatitis viruses (VSV) expressing foreign viral proteins are protective in several animal disease models. Although these attenuated viruses are nonpathogenic in nonhuman primates when given by nasal, oral, or intramuscular routes, they are pathogenic in mice when given intranasally, and further vector attenuation may be required before human trials with VSV-based vectors can begin. Mutations truncating the VSV glycoprotein (G) cytoplasmic domain from 29 to 9 or 1 amino acid (designated CT9 or CT1, respectively) were shown previously to attenuate VSV growth in cell culture and pathogenesis in mice. Here we show that VSV recombinants carrying either the CT1 or CT9 deletion and expressing the human immunodeficiency virus (HIV) Env protein are nonpathogenic in mice, even when given by the intranasal route. We then carried out a detailed analysis of the CD8+T-cell responses, including in vivo cytotoxic T-cell activity, induced by these vectors. When given by either the intranasal or intraperitoneal route, the VSV-CT9 vector expressing HIV Env elicited primary and memory CD8+T-cell responses to Env equivalent to those elicited by recombinant wild-type VSV expressing Env. The VSV-CT1 vector also induced potent CD8+T-cell responses after intraperitoneal vaccination, but was less effective when given by the intranasal route. The VSV-CT1 vector was also substantially less effective than the VSV-CT9 or wild-type vector at inducing antibody to Env. The VSV-CT9 vector appears ideal because of its lack of pathogenesis, propagation to high titers in vitro, and stimulation of strong cellular and humoral immune responses.
Expression of the Two Nested Overlapping Reading Frames of Turnip Yellow Mosaic Virus RNA Is Enhanced by a 5′ Cap and by 5′ and 3′ Viral Sequences
Authors: Matsuda Daiki , Bauer Lisa , Tinnesand Kathryn , Dreher Theo W.
Published: August 2004

The translation efficiency of an mRNA molecule is typically determined by its 5′- and/or 3′-untranslated regions (UTRs). Previously, we have found that the 3′-UTR of
Turnip yellow mosaic virus
(TYMV) RNA enhances translation synergistically with a 5′ cap. Here, we use a luciferase reporter system in cowpea protoplasts to show that the 5′ 217 nucleotides from TYMV genomic RNA enhance expression relative to a vector-derived 17-nucleotide 5′-UTR. Maximum expression was observed from RNAs with a cap and both 5′ and 3′ TYMV sequences. In paired reporter constructs, the 5′ 217 nucleotides harboring the UTR and the first 43 or 41 codons of the two overlapping TYMV open reading frames (ORFs), ORF-69 and ORF-206, respectively, were fused in frame with the luciferase gene. This allowed expression from the initiation codon of each ORF (AUG
69
and AUG
206
) to be monitored separately but from the normal sequence environment. Expression from both AUG codons was heavily dependent on a 5′ cap, with a threefold-higher expression occurring from AUG
69
than from AUG
206
in the presence of the genomic 3′-UTR. Changes that interrupted the cap/3′-UTR synergy (i.e., removal of the cap or TYMV 3′-UTR) resulted in a higher proportion of initiation from AUG
206
. Mutation of the 3′-UTR to prevent aminoacylation, as well as deletion of 75% of the 5′-UTR, likewise resulted in a lower ratio of expression from AUG
69
relative to AUG
206
. Mutation of each AUG initiation codon increased initiation from the other. Taken together, these results do not fully conform to the expectations of standard leaky ribosomal scanning and leave open the precise mechanism of ribosome commitment to AUG
69
and AUG
206
. However, our observations do not support a recent proposal based on in vitro studies in which the 3′-UTR is proposed to direct cap-independent initiation specifically at AUG
206
and not at AUG
69
(S. Barends et al., Cell 112:123-129, 2003).
Tumorigenesis by Human Herpesvirus 8 vGPCR Is Accelerated by Human Immuodeficiency Virus Type 1 Tat
Authors: Guo Hong-Guang , Pati Shibani , Sadowska Mariola , Charurat Man , Reitz Marvin
Published: August 2004

Human herpesvirus 8 (HHV-8), also called Kaposi's sarcoma (KS) herpesvirus, can cause KS but is inefficient. Untreated human immunodeficiency virus type 1 (HIV-1) coinfection is a powerful risk factor. The HHV-8 chemokine receptor, vGPCR (ORF74), activates NF-κB and NF-AT, and their levels of activation are synergistically increased by HIV-1 Tat. Transgenic vGPCR mice develop KS-like tumors. A cell line derived from one such tumor expresses vGPCR and forms tumors in nude mice. Here we show that transfection of DNA encoding HIV-1tat(but not a transactivation-defective mutant) into these tumor cells increases NF-κB and NF-AT activation levels and accelerates tumor formation. Tumorigenesis was also accelerated when Tat DNA was transfected into normal cells and the transfected cells were mixed with the tumor cells and injected into a single site. Tumorigenesis was also increased when the two cell types were injected at separate sites, suggesting that tumorigenesis is accelerated by Tat through soluble factors.
Roscovitine Inhibits Activation of Promoters in Herpes Simplex Virus Type 1 Genomes Independently of Promoter-Specific Factors
Authors: Diwan Prerna , Lacasse Jonathan J. , Schang Luis M.
Published: August 2004

Flavopiridol, roscovitine, and other inhibitors of Cyclin-Dependent Kinases (CDK) inhibit the replication of a variety of viruses in vitro while proving nontoxic in human clinical trials of their effects against cancer. Consequently, these and other Pharmacological CDK inhibitors (PCIs) have been proposed as potential antivirals. Flavopiridol potently inhibits all tested CDKs and inhibits the transcription of most cellular and viral genes. In contrast, roscovitine and other purine PCIs inhibit with high potency only CDK1, CDK2, CDK5, and CDK7, and they specifically inhibit the expression of viral but not cellular genes. The levels at which purine PCIs inhibit gene expression are unknown, as are the factors which determine their specificity for expression of viral but not cellular genes. We show herein that roscovitine prevents the initiation of transcription of herpes simplex virus type 1 (HSV-1) genes but has no effect on transcription elongation. We further show that roscovitine does not inhibit the initiation or elongation of cellular transcription and that its inhibitory effects are specific for promoters in HSV-1 genomes. Therefore, we have identified a novel biological activity for PCIs, i.e., their ability to prevent the initiation of transcription. We have also identified genome location as one of the factors that determine whether the transcription of a given gene is inhibited by roscovitine. The activities of roscovitine on viral transcription resemble one of the antiherpesvirus activities of alpha interferon and could be used as a model for the development of novel antivirals. The genome-specific effects of roscovitine may also be important for its development against virus-induced cancers.
In Vivo Analysis of the 3′ Untranslated Region of GB Virus B after In Vitro Mutagenesis of an Infectious cDNA Clone: Persistent Infection in a Transfected Tamarin
Authors: Nam Jae-Hwan , Faulk Kristina , Engle Ronald E. , Govindarajan Sugantha , St. Claire Marisa , Bukh Jens
Published: August 2004

GB virus B (GBV-B), the virus most closely related to hepatitis C virus (HCV), infects tamarins and causes acute hepatitis. The 3′ untranslated region (UTR) of an infectious GBV-B clone (pGBB) has a proximal short sequence followed by a poly(U) tract and a 3′ terminal sequence. Our investigators previously demonstrated that the 3′ terminal sequence was critical for in vivo infectivity. Here, we tested the effect of deleting the short sequence and/or the poly(U) tract from pGBB; infectivity of each mutant was tested by intrahepatic transfection of two tamarins with transcribed RNA. A mutant lacking both regions was not viable. However, mutants lacking either the short sequence or the poly(U) tract were viable. All four tamarins had a wild-type-like acute infection and developed acute hepatitis. Whereas we found that five tamarins transfected with the wild-type clone pGBB had acute resolving infection, one tamarin transfected with the poly(U) deletion mutant became persistently infected. This animal had viremia and hepatitis until its death at week 90. The genomes recovered at weeks 2, 7, 15, 20, 60, and 90 lacked the poly(U) stretch. Eight amino acid changes were identified at week 90. One change, in the putative p7 protein, was dominant at week 15. Thus, persistence of GBV-B, like persistence of HCV, was associated with the emergence of virus variants. Four tamarins inoculated with serum collected at weeks 2 and 90 from the tamarin with persistent infection had an acute resolving infection. Nonetheless, the demonstration that GBV-B can persist in tamarins strengthens its relevance as a surrogate model for the study of HCV.
Immunomodulatory Effects of Inactivated Parapoxvirus Ovis (Orf Virus) on Human Peripheral Immune Cells: Induction of Cytokine Secretion in Monocytes and Th1-Like Cells
Authors: Friebe Astrid , Siegling Angela , Friederichs Sonja , Volk Hans-Dieter , Weber Olaf
Published: August 2004

Inactivated parapoxvirus ovis (Orf virus; PPVO) recently displayed strong immunostimulating and modulating capacities in several animal models for acute and chronic virus infections through the induction of gamma interferon (IFN-γ) as a key mediator of antiviral activity. The data presented in this work demonstrate that inactivated PPVO has strong effects on cytokine secretion by human immune cells, including the upregulation of inflammatory and Th1-related cytokines (IFN-γ, tumor necrosis factor alpha [TNF-α], interleukin 6 [IL-6], IL-8, IL-12, and IL-18) as well as anti-inflammatory and Th2-related cytokines (IL-4, IL-10, and IL-1 receptor antagonist [IL-1ra]). Studies on the mechanism of action revealed virus particles to be the effective components of the preparation. The virus particles activate monocytes or other antigen-presenting cells (APC), e.g., plasmacytoid dendritic cells, through signaling over CD14 and a Toll-like receptor and the intracellular presence of certain PPVO-specific components. The activation of monocytes or APC is followed by the release of early proinflammatory cytokines (TNF-α, IL-6, and IL-8) as well as the Th1-related cytokines IL-12 and IL-18. Both IL-18 and IL-12 are involved in PPVO-mediated IFN-γ release by T cells and/or NK cells. The proinflammatory response is accompanied by the induction of anti-inflammatory and Th2-related cytokines (IL-4, IL-10, and IL-1ra), which exert a limiting efffect on the inflammatory response induced by PPVO. We conclude that the induction of a natural immune response with physiologically significant amounts of different cytokines and with antiviral potential might provide advantages over existing antiviral immunotherapies.
Role of the C Terminus of Foamy Virus Gag in RNA Packaging and Pol Expression
Authors: Stenbak Carolyn R. , Linial Maxine L.
Published: August 2004

Foamy viruses (FV) are complex retroviruses that possess several unique features that distinguish them from all other retroviruses. FV Gag and Pol proteins are expressed independently of one another, and both proteins undergo single cleavage events. Thus, the mature FV Gag protein does not consist of the matrix, capsid, and nucleocapsid (NC) proteins found in orthoretroviruses, and the putative NC domain of FV Gag lacks the hallmark Cys-His motifs or I domains. As there is no Gag-Pol fusion protein, the mechanism of Pol packaging is different but unknown. FV RNA packaging is not well understood either. The C terminus of FV Gag has three glycine-arginine motifs (GR boxes), the first of which has been shown to have nucleic acid binding properties in vitro. The role of these GR boxes in RNA packaging and Pol packaging was investigated with a series of Gag C-terminal truncation mutants. GR box 1 was found to be the major determinant of RNA packaging, but all three GR boxes were required to achieve wild-type levels of RNA packaging. In addition, Pol was packaged in the absence of GR box 3, but GR boxes 1 and 2 were required for efficient Pol packaging. Interestingly, the Gag truncation mutants demonstrated decreased Pol expression levels as well as defects in Pol cleavage. Thus, the C terminus of FV Gag was found to be responsible for RNA packaging, as well as being involved in the expression, cleavage, and incorporation of the Pol protein.
Host Cell Specificity of Minute Virus of Mice in the Developing Mouse Embryo
Authors: Itah Refael , Tal Jacov , Davis Claytus
Published: August 2004

Productive infection by the murine autonomous parvovirus minute virus of mice (MVM) depends on a dividing cell population and its differentiation state. We have extended the in vivo analysis of the MVM host cell type range into the developing embryo by in utero inoculation followed by further gestation. The fibrotropic p strain (MVMp) and the lymphotropic i strain (MVMi) did not productively infect the early mouse embryo but were able to infect overlapping sets of cell types in the mid- or late-gestation embryo. Both MVMp and MVMi infected developing bone primordia, notochord, central nervous system, and dorsal root ganglia. MVMp exhibited extensive infection in fibroblasts, in the epithelia of lung and developing nose, and, to a lesser extent, in the gut. MVMi also infected endothelium. The data indicated that the host ranges of the two MVM strains consist of overlapping sets of cell types that are broader than previously known from neonate and in vitro infection experiments. The correlation between MVM host cell types and the cell types that activate the transgenic P4 promoter is consistent with the hypothesis that activation of the incoming viral P4 promoter by the host cell is one of the host range determinants of MVM.
LEDGF/p75 Determines Cellular Trafficking of Diverse Lentiviral but Not Murine Oncoretroviral Integrase Proteins and Is a Component of Functional Lentiviral Preintegration Complexes
Authors: Llano Manuel , Vanegas Maria , Fregoso Oliver , Saenz Dyana , Chung Susan , Peretz Mary , Poeschla Eric M.
Published: August 2004

Human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and Moloney murine leukemia virus (MoMLV) integrases were stably expressed to determine their intracellular trafficking. Each lentiviral integrase localized to cell nuclei in close association with chromatin while the murine oncoretroviral integrase was cytoplasmic. Fusions of pyruvate kinase to the lentiviral integrases did not reveal transferable nuclear localization signals. The intracellular trafficking of each was determined instead by the transcriptional coactivator LEDGF/p75, which was required for nuclear localization. Stable small interfering RNA expression eliminated detectable LEDGF/p75 expression and caused dramatic, stable redistribution of each lentiviral integrase from nucleus to cytoplasm while the distribution of MoMLV integrase was unaffected. In addition, endogenous LEDGF/p75 coimmunoprecipitated specifically with each lentiviral integrase. In vitro integration assays with preintegration complexes (PICs) showed that endogenous LEDGF/p75 is a component of functional HIV-1 and FIV PICs. However, HIV-1 and FIV infection and replication in LEDGF/p75-deficient cells was equivalent to that in control cells, whether cells were dividing or growth arrested. Two-long terminal repeat circle accumulation in nondividing cell nuclei was also equivalent to that of LEDGF/p75 wild-type cells. Virions produced in LEDGF/p75-deficient cells had normal infectivity. We conclude that LEDGF/p75 fully accounts for cellular trafficking of diverse lentiviral, but not oncoretroviral, integrases and is the main lentiviral integrase-to-chromatin tethering factor. While lentiviral PIC nuclear import is unaffected by LEDGF/p75 knockdown, this protein is a component of functional lentiviral PICs. A role in HIV-1 integration site distribution merits investigation.
Hepatitis B Virus Capsid Assembly Is Enhanced by Naturally Occurring Mutation F97L
Authors: Ceres Pablo , Stray Stephen J. , Zlotnick Adam
Published: August 2004

In chronic hepatitis B virus (HBV) infections, one of the most common mutations to the virus occurs at amino acid 97 of the core protein, where leucine replaces either phenylalanine or isoleucine, depending on strain. This mutation correlates with changes in viral nucleic acid metabolism and/or secretion. We hypothesize that this phenotype is due in part to altered core assembly, a process required for DNA synthesis. We examined in vitro assembly of empty HBV capsids from wild-type and F97L core protein assembly domains. The mutation enhanced both the rate and extent of assembly relative to those for the wild-type protein. The difference between the two proteins was most obvious in the temperature dependence of assembly, which was dramatically stronger for the mutant protein, indicating a much more positive enthalpy. Since the structures of the mutant and wild-type capsids are essentially the same and the mutation is not involved in the contact between dimers, we suggest that the F97L mutation affects the dynamic behavior of dimer and capsid.
Measles Virus Interacts with and Alters Signal Transduction in T-Cell Lipid Rafts
Authors: Avota Elita , Müller Nora , Klett Maren , Schneider-Schaulies Sibylle
Published: August 2004

By a contact-dependent surface interaction, the measles virus (MV) glycoprotein complex induces a pronounced inhibition of T-cell proliferation. We now show that MV directly interacts with glycosphingolipid-enriched membrane microdomains on human primary T cells and alters recruitment and segregation of membrane proximal signaling components. Contact-dependent interference with T-cell receptor-stimulated tyrosine phosphorylation and Ca mobilization is a late event seen 24 h after MV treatment. In contrast, stimulated recruitment of pleckstrin homology domain-containing proteins such as Akt and Vav is inhibited early after MV contact, as is segregation of the activated Akt kinase from rafts. Tyrosine phosphorylation of the regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), p85, is apparently normal then, yet this protein fails to partition to the lipid raft fraction, and this is associated with stable expression of its negative regulator Cbl-b. Thus, by interaction with lipid rafts, MV contact initially targets recruitment of PI3K by preventing stimulated Cbl-b degradation and activation of PI3K-dependent signaling components.
Characterization of the Human Cytomegalovirus UL34 Gene
Authors: Biegalke B. J. , Lester E. , Branda A. , Rana R.
Published: August 2004

UL34 encodes the transcriptional repressor of the human cytomegalovirus immune evasion gene, US3, and is essential for viral replication in tissue culture. Two different monocistronic transcripts originate from UL34 at early and late times postinfection and encode two predominant proteins and a third, minor protein. The UL34 proteins are differentially expressed throughout the viral replication cycle, with both proteins localizing to the nucleus and repressing expression of the US3 gene.