bluuebunny/crossref_metadata_2025 huggingface dataset:
A Single-Cycle Vaccine Vector Based on Vesicular Stomatitis Virus Can Induce Immune Responses Comparable to Those Generated by a Replication-Competent Vector
Authors: Publicover Jean , Ramsburg Elizabeth , Rose John K.
Published: October 2005

Live attenuated vaccine vectors based on recombinant vesicular stomatitis virus (VSV) are effective in several viral disease models. In this study, we asked if a VSV vector capable of only a single cycle of replication might be an effective alternative to replication-competent VSV vectors. We compared the cellular immune responses to human immunodeficiency virus (HIV) envelope protein (Env) expressed by replication-competent and single-cycle VSV vectors and also examined the antibody response to Env. The single-cycle vector was grown by complementation with VSV G protein and then tested initially for immunogenicity when given by four different routes. When given by the intramuscular route in mice, we found that the single-cycle vector was equivalent to the replication-competent VSV vector in generating high-level primary and memory CD8 T-cell responses as well as antibody responses to Env. Cellular responses were analyzed using major histocompatibility complex class I tetramers and direct measurement of cytotoxic T-lymphocyte activity in vivo. We also found that the recall responses after boosting were equivalent in animals vaccinated with replication-competent or single-cycle vectors. Additionally, we observed recall and heightened memory responses after boosting animals with a single-cycle vector complemented with G protein from a different vesiculovirus. Because expression of HIV Env by G-deleted VSV might allow replication in human cells expressing CD4, we generated a single-cycle VSV recombinant expressing a secreted form of the HIV Env protein. This virus was just as effective as the recombinant expressing the membrane-anchored Env protein at producing CD8 T cells and antibody responses.
Selective Escape from CD8
+
T-Cell Responses Represents a Major Driving Force of Human Immunodeficiency Virus Type 1 (HIV-1) Sequence Diversity and Reveals Constraints on HIV-1 Evolution
Authors: Allen Todd M. , Altfeld Marcus , Geer Shaun C. , Kalife Elizabeth T. , Moore Corey , O'Sullivan Kristin M. , DeSouza Ivna , Feeney Margaret E. , Eldridge Robert L. , Maier Erica L. , Kaufmann Daniel E. , Lahaie Matthew P. , Reyor Laura , Tanzi Giancarlo , Johnston Mary N. , Brander Christian , Draenert Rika , Rockstroh Jurgen K. , Jessen Heiko , Rosenberg Eric S. , Mallal Simon A. , Walker Bruce D.
Published: October 2005

The sequence diversity of human immunodeficiency virus type 1 (HIV-1) represents a major obstacle to the development of an effective vaccine, yet the forces impacting the evolution of this pathogen remain unclear. To address this issue we assessed the relationship between genome-wide viral evolution and adaptive CD8
+
T-cell responses in four clade B virus-infected patients studied longitudinally for as long as 5 years after acute infection. Of the 98 amino acid mutations identified in nonenvelope antigens, 53% were associated with detectable CD8
+
T-cell responses, indicative of positive selective immune pressures. An additional 18% of amino acid mutations represented substitutions toward common clade B consensus sequence residues, nine of which were strongly associated with HLA class I alleles not expressed by the subjects and thus indicative of reversions of transmitted CD8 escape mutations. Thus, nearly two-thirds of all mutations were attributable to CD8
+
T-cell selective pressures. A closer examination of CD8 escape mutations in additional persons with chronic disease indicated that not only did immune pressures frequently result in selection of identical amino acid substitutions in mutating epitopes, but mutating residues also correlated with highly polymorphic sites in both clade B and C viruses. These data indicate a dominant role for cellular immune selective pressures in driving both individual and global HIV-1 evolution. The stereotypic nature of acquired mutations provides support for biochemical constraints limiting HIV-1 evolution and for the impact of CD8 escape mutations on viral fitness.
A Redox-Sensitive Cysteine in Zta Is Required for Epstein-Barr Virus Lytic Cycle DNA Replication
Authors: Wang Pu , Day Latasha , Dheekollu Jayaraju , Lieberman Paul M.
Published: October 2005

Epstein-Barr virus (EBV) reactivation from latency is known to be sensitive to redox regulation. The immediate-early protein Zta is a member of the basic-leucine zipper (bZIP) family of DNA binding proteins that stimulates viral and cellular transcription and nucleates a replication complex at the viral lytic origin. Zta shares with several members of the bZIP family a conserved cysteine residue (C189) that confers redox regulation of DNA binding. In this work, we show that replacement of C189 with serine (C189S) eliminated lytic cycle DNA replication function of Zta. The mechanistic basis for this replication defect was investigated. We show that C189S was not significantly altered for DNA binding activity in vitro or in vivo. We also show that C189S was not defective for transcription activation of EBV early gene promoters. C189S was deficient for transcription activation of several viral late genes that depend on lytic replication and therefore was consistent with a primary defect of C189S in activating lytic replication. C189S was not defective in binding methylated DNA binding sites and was capable of activating Rta from endogenous latent viral genomes, in contrast to the previously characterized S186A mutation. C189S was slightly impaired for its ability to form a stable complex with Rta, although this did not prevent Rta recruitment to OriLyt. C189S did provide some resistance to oxidation and nitrosylation, which potently inhibit Zta DNA binding activity in vitro. Interestingly, this redox sensitivity was not strictly dependent on C189S but involved additional cysteine residues in Zta. These results provide evidence that the conserved cysteine in the bZIP domain of Zta plays a primary role in EBV lytic cycle DNA replication.
Evolution of Hepatitis Delta Virus RNA Genome following Long-Term Replication in Cell Culture
Authors: Chang Jinhong , Gudima Severin O. , Taylor John M.
Published: October 2005

Previous studies have defined a novel cell culture system in which a modified RNA genome of hepatitis delta virus (HDV) is able to maintain a low level of continuous replication for at least 1 year, using a separate and limited DNA-directed source of mRNA for the essential small delta protein. This mode of replication is analogous to that used by plant viroids. An examination was made of the nucleotide changes that accumulated on the HDV RNA during 1 year of replication. The length of the RNA genome was maintained, except for some single-nucleotide deletions and insertions. There was an abundance of single-nucleotide substitutions, with a 22-fold excess of these being base transitions rather than transversions. Of the detected transitions, at least 70% were consistent with being the consequences of posttranscriptional RNA editing by an adenosine deaminase acting on RNA. The remainder of the changes, including the single-nucleotide insertions and deletions, are likely to be the consequence of misincorporation during transcription. In addition, an intermolecular competition assay was used to show that the majority of the genomes present after 1 year of replication were essentially as competent in replication as the original single HDV RNA sequence that was used to initiate the genome replication. A model is provided to explain how, in this experimental system, the observed single-nucleotide changes were essentially neutral in terms of their effect on the ability of the HDV genome to carry out continued rounds of replication.
The Endoplasmic Reticulum Lumenal Domain of the Adenovirus Type 2 E3-19K Protein Binds to Peptide-Filled and Peptide-Deficient HLA-A*1101 Molecules
Authors: Liu Hong , Stafford Walter F. , Bouvier Marlene
Published: October 2005

E3-19K is a type I membrane glycoprotein expressed by adenoviruses (Ads) to modulate host antiviral immune responses. We have developed an expression system for the endoplasmic reticulum lumenal domain (residues 1 to 100) of Ad type 2 E3-19K tagged with a C-terminal His6sequence in baculovirus-infected insect cells. In this system, recombinant E3-19K is secreted into the culture medium. A characterization of soluble E3-19K by analytical ultracentrifugation and circular dichroism showed that the protein is monomeric and adopts a stable and correctly folded tertiary structure. Using a gel mobility shift assay and analytical ultracentrifugation, we showed that soluble E3-19K associates with soluble peptide-filled and peptide-deficient HLA-A*1101 molecules. This is the first example of a viral immunomodulatory protein that interacts with conformationally distinct forms of class I major histocompatibility complex molecules. The E3-19K/HLA-A*1101 complexes formed in a 1:1 stoichiometry with equilibrium dissociation constants (Kd) of 50 ± 10 nM for peptide-filled molecules and of about 10 μM for peptide-deficient molecules. A temperature-dependent proteolysis study revealed that the association of E3-19K with peptide-deficient HLA-A*1101 molecules stabilizes the binding groove. Importantly, our studies showed that peptide-deficient HLA-A*1101 molecules sequestered by E3-19K are capable of binding antigenic peptides and maturing into peptide-filled molecules. This firmly establishes that E3-19K does not block binding of antigenic peptides. Together, our results suggest that Ads have evolved to exploit the late and early stages of the class I antigen presentation pathway.
Evaluation of Envelope Vaccines Derived from the South African Subtype C Human Immunodeficiency Virus Type 1 TV1 Strain
Authors: Lian Ying , Srivastava Indresh , Gómez-Román V. Rául , zur Megede Jan , Sun Yide , Kan Elaine , Hilt Susan , Engelbrecht Susan , Himathongkham Sunee , Luciw Paul A. , Otten Gillis , Ulmer Jeffrey B. , Donnelly John J. , Rabussay Dietmar , Montefiori David , van Rensburg Estrelita Janse , Barnett Susan W.
Published: October 2005

Human immunodeficiency virus type 1 (HIV-1) subtype C infections are on the rise in Sub-Saharan Africa and Asia. Therefore, there is a need to develop an HIV vaccine capable of eliciting broadly reactive immune responses against members of this subtype. We show here that modified HIV envelope (env) DNA vaccines derived from the South African subtype C TV1 strain are able to prime for humoral responses in rabbits and rhesus macaques. Priming rabbits with DNA plasmids encoding V2-deleted TV1 gp140 (gp140TV1ΔV2), followed by boosting with oligomeric protein (o-gp140TV1ΔV2) in MF59 adjuvant, elicited higher titers of env-binding and autologous neutralizing antibodies than priming with DNA vaccines encoding the full-length TV1 env (gp160) or the intact TV1 gp140. Immunization with V2-deleted subtype B SF162 env and V2-deleted TV1 env together using a multivalent vaccine approach induced high titers of oligomeric env-binding antibodies and autologous neutralizing antibodies against both the subtypes B and C vaccine strains, HIV-1 SF162 and TV1, respectively. Low-level neutralizing activity against the heterologous South African subtype C TV2 strain, as well as a small subset of viruses in a panel of 13 heterologous primary isolates, was observed in some rabbits immunized with the V2-deleted vaccines. Immunization of rhesus macaques with the V2-deleted TV1 DNA prime/protein boost also elicited high titers of env-binding antibodies and moderate titers of autologous TV1 neutralizing antibodies. The pilot-scale production of the various TV1 DNA vaccine constructs and env proteins described here should provide an initial platform upon which to improve the immunogenicity of these subtype C HIV envelope vaccines.
Herpes Simplex Virus Type 1 Glycoprotein E Is Required for Axonal Localization of Capsid, Tegument, and Membrane Glycoproteins
Authors: Wang Fushan , Tang Waixing , McGraw Helen M. , Bennett Jean , Enquist Lynn W. , Friedman Harvey M.
Published: October 2005

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) promotes cell-to-cell spread at basolateral surfaces of epithelial cells, but its activity in neurons is less clear. We used the mouse retina infection model and neuronal cell cultures to define the spread phenotype of gE mutant viruses. Wild-type (WT) and gE-null (NS-gEnull) viruses both infected retina ganglion cell neurons; however, NS-gEnull viral antigens failed to reach the optic nerve, which indicates a defect in axonal localization. We evaluated two Fc receptor-negative gE mutant viruses containing four amino acid inserts in the gE ectodomain. One mutant virus failed to spread from the retina into the optic nerve, while the other spread normally. Therefore, the gE ectodomain is involved in axonal localization, and the Fc receptor and neuronal spread are mediated by overlapping but distinct gE domains. In the retina infection model, virus can travel to the brain via the optic nerve from presynaptic to postsynaptic neurons (anterograde direction) or via nerves that innervate the iris and ciliary body from postsynaptic to presynaptic neurons (retrograde direction). WT virus infected the brain by anterograde and retrograde routes, whereas NS-gEnull virus failed to travel by either pathway. The site of the defect in retrograde spread remains to be determined; however, infection of rat superior cervical ganglia neurons in vitro indicates that gE is required to target virion components to the axon initial segment. The requirement for gE in axonal targeting and retrograde spread highlights intriguing similarities and differences between HSV-1 and pseudorabies virus gE.
Amino Acid Residues within Conserved Domain VI of the Vesicular Stomatitis Virus Large Polymerase Protein Essential for mRNA Cap Methyltransferase Activity
Authors: Li Jianrong , Fontaine-Rodriguez Errin C. , Whelan Sean P. J.
Published: October 2005

During mRNA synthesis, the polymerase of vesicular stomatitis virus (VSV) copies the genomic RNA to produce five capped and polyadenylated mRNAs with the 5′-terminal structure 7
m
GpppA
m
pApCpApGpNpNpApUpCp. The 5′ mRNA processing events are poorly understood but presumably require triphosphatase, guanylyltransferase, [guanine-N-7]- and [ribose-2′-O]-methyltransferase (MTase) activities. Consistent with a role in mRNA methylation, conserved domain VI of the 241-kDa large (L) polymerase protein shares sequence homology with a bacterial [ribose-2′-O]-MTase, FtsJ/RrmJ. In this report, we generated six
L
gene mutations to test this homology. Individual substitutions to the predicted MTase active-site residues K1651, D1762, K1795, and E1833 yielded viruses with pinpoint plaque morphologies and 10- to 1,000-fold replication defects in single-step growth assays. Consistent with these defects, viral RNA and protein synthesis was diminished. In contrast, alteration of residue G1674 predicted to bind the methyl donor
S
-adenosylmethionine did not significantly perturb viral growth and gene expression. Analysis of the mRNA cap structure revealed that alterations to the predicted active site residues decreased [guanine-N-7]- and [ribose-2′-O]-MTase activity below the limit of detection of our assay. In contrast, the alanine substitution at G1674 had no apparent consequence. These data show that the predicted MTase active-site residues K1651, D1762, K1795, and E1833 within domain VI of the VSV L protein are essential for mRNA cap methylation. A model of mRNA processing consistent with these data is presented.
Identification, Subviral Localization, and Functional Characterization of the Pseudorabies Virus UL17 Protein
Authors: Klupp Barbara G. , Granzow Harald , Karger Axel , Mettenleiter Thomas C.
Published: October 2005

Homologs of the UL17 gene of the alphaherpesvirus herpes simplex virus 1 (HSV-1) are conserved in all three subfamilies of herpesviruses. However, only the HSV-1 protein has so far been characterized in any detail. To analyze UL17 of pseudorabies virus (PrV) the complete 597-amino-acid protein was expressed in
Escherichia coli
and used for rabbit immunization. The antiserum recognized a 64-kDa protein in PrV-infected cell lysates and purified virions, identifying PrV UL17 as a structural virion component. In indirect immunofluorescence analyses of PrV-infected cells the protein was predominantly found in the nucleus. In electron microscopic studies after immunogold labeling of negatively stained purified virion preparations, UL17-specific label was detected on single, mostly damaged capsids, whereas complete virions and the majority of capsids were free of label. In ultrathin sections of infected cells, label was primarily found dispersed around scaffold-containing B-capsids, whereas on DNA-filled C-capsids it was located in the center. Empty intranuclear A-capsids were free of label, as were extracellular capsid-less L-particles. Functional characterization of PrV-ΔUL17F, a deletion mutant lacking codons 23 to 444, demonstrated that cleavage of viral DNA into unit-length genomes was inhibited in the absence of UL17. In electron microscopic analyses of PrV-ΔUL17F-infected RK13 cells, DNA-containing capsids were not detected, while numerous capsidless L-particles were observed. In summary, our data indicate that the PrV UL17 protein is an internal nucleocapsid protein necessary for DNA cleavage and packaging but suggest that the protein is not a prominent part of the tegument.
Combinations of Polyclonal or Monoclonal Antibodies to Proteins of the Outer Membranes of the Two Infectious Forms of Vaccinia Virus Protect Mice against a Lethal Respiratory Challenge
Authors: Lustig Shlomo , Fogg Christiana , Whitbeck J. Charles , Eisenberg Roselyn J. , Cohen Gary H. , Moss Bernard
Published: October 2005

Previous studies demonstrated that antibodies to live vaccinia virus infection are needed for optimal protection against orthopoxvirus infection. The present report is the first to compare the protective abilities of individual and combinations of specific polyclonal and monoclonal antibodies that target proteins of the intracellular (IMV) and extracellular (EV) forms of vaccinia virus. The antibodies were directed to one IMV membrane protein, L1, and to two outer EV membrane proteins, A33 and B5. In vitro studies showed that the antibodies to L1 neutralized IMV and that the antibodies to A33 and B5 prevented the spread of EV in liquid medium. Prophylactic administration of individual antibodies to BALB/c mice partially protected them against disease following intranasal challenge with lethal doses of vaccinia virus. Combinations of antibodies, particularly anti-L1 and -A33 or -L1 and -B5, provided enhanced protection when administered 1 day before or 2 days after challenge. Furthermore, the protection was superior to that achieved with pooled immune gamma globulin from human volunteers inoculated with live vaccinia virus. In addition, single injections of anti-L1 plus anti-A33 antibodies greatly delayed the deaths of severe combined immunodeficiency mice challenged with vaccinia virus. These studies suggest that antibodies to two or three viral membrane proteins optimally derived from the outer membranes of IMV and EV, may be beneficial for prophylaxis or therapy of orthopoxvirus infections.
Human VAP-B Is Involved in Hepatitis C Virus Replication through Interaction with NS5A and NS5B
Authors: Hamamoto Itsuki , Nishimura Yorihiro , Okamoto Toru , Aizaki Hideki , Liu Minyi , Mori Yoshio , Abe Takayuki , Suzuki Tetsuro , Lai Michael M. C. , Miyamura Tatsuo , Moriishi Kohji , Matsuura Yoshiharu
Published: October 2005

The hepatitis C virus (HCV) nonstructural protein (NS) 5A is a phosphoprotein that associates with various cellular proteins and participates in the replication of the HCV genome. Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) is known to be a host factor essential for HCV replication by binding to both NS5A and NS5B. To obtain more information on the NS5A protein in HCV replication, we screened human brain and liver libraries by a yeast two-hybrid system using NS5A as bait and identified VAP-B as an NS5A-binding protein. Immunoprecipitation and mutation analyses revealed that VAP-B binds to both NS5A and NS5B in mammalian cells and forms homo- and heterodimers with VAP-A. VAP-A interacts with VAP-B through the transmembrane domain. NS5A interacts with the coiled-coil domain of VAP-B via 70 residues in the N-terminal and 341 to 344 amino acids in the C-terminal polyproline cluster region. NS5A was colocalized with VAP-B in the endoplasmic reticulum and Golgi apparatus. The specific antibody to VAP-B suppressed HCV RNA replication in a cell-free assay. Overexpression of VAP-B, but not of a mutant lacking its transmembrane domain, enhanced the expression of NS5A and NS5B and the replication of HCV RNA in Huh-7 cells harboring a subgenomic replicon. In the HCV replicon cells, the knockdown of endogenous VAP-B by small interfering RNA decreased expression of NS5B, but not of NS5A. These results suggest that VAP-B, in addition to VAP-A, plays an important role in the replication of the HCV genome.
Reduced Secretion of Virions and Hepatitis B Virus (HBV) Surface Antigen of a Naturally Occurring HBV Variant Correlates with the Accumulation of the Small S Envelope Protein in the Endoplasmic Reticulum and Golgi Apparatus
Authors: Chua Pong Kian , Wang Robert Yung-Liang , Lin Min-Hui , Masuda Tetsuya , Suk Fat-Moon , Shih Chiaho
Published: October 2005

We identified two novel naturally occurring mutations (W74L and L77R) in the small S envelope protein of hepatitis B virus (HBV). Mutation L77R alone resulted in >10-fold-reduced secretion of virions. In addition, the 2.8-fold reduction of the extracellular HBV surface antigen (HBsAg) of mutant L77R from transfected Huh7 cells appeared to be correlated with a 1.7-fold reduction of intracellular HBsAg, as measured by enzyme-linked immunosorbent assay (ELISA). Surprisingly, opposite to the ELISA results, Western blot analysis revealed a near-10-fold-increased level of the intracellular mutant small S envelope protein. The discrepancy between ELISA and Western blot data was due to significant accumulation of the mutant L77R HBsAg in the intracellular pellet fraction. In contrast to HBsAg, the secretion of HBeAg was normal in L77R-transfected cells. The wild-type HBsAg was usually more diffuse and evenly distributed in the cytoplasm, often outside the perinuclear endoplasmic reticulum (ER) and Golgi apparatus, as observed by immunofluorescence assay. In contrast, the L77R mutant HBsAg tends to be highly restricted within the ER and Golgi, often accumulated in the Golgi compartments distal from the nucleus. The almost exclusive retention in the ER-Golgi of L77R HBsAg was similar to what was observed when the large envelope protein was overexpressed. These multiple aberrant phenotypes of mutant L77R can be corrected by a second naturally occurring S envelope mutation, W74L. Despite the accumulation of L77R HBsAg in ER-Golgi of transfected Huh7 cells, we detected no increase in Grp78 mRNA and proteins, which are common markers for ER stress response.
Dynamic DNA Methylation and Histone Modifications Contribute to Lentiviral Transgene Silencing in Murine Embryonic Carcinoma Cells
Authors: He Jin , Yang Qing , Chang Lung-Ji
Published: October 2005

Embryonic stem cells are subjected to a dynamic genome regulation during development. Here we report that the ectopic lentiviral transgenes are quickly silenced in murine embryonic carcinoma P19 cells. The silencing was correlated with CpG hypermethylation in the transgene promoter. Using high-resolution sodium bisulfite genome sequencing, we detected distinct DNA methylation kinetics in different proviral regions. DNase I sensitivity and chromatin immunoprecipitation assays revealed condensed chromatin structure and histone code switch during silencing. Longitudinal analysis of nonsilenced and silenced identical single-cell clones revealed that the silencing was coupled with CpG methylation in the promoter, as well as a global histone H3 deacetylation. Interestingly, the primer binding site and the packaging signal region appeared to serve as a DNA methylation initiation center which was rapidly hypermethylated regardless of transgene silencing and chromatin modifications. Analysis of cellular genes 45 to 50 kbp upstream and downstream of the integration site indicated that transcriptional activities of the flanking host genes were not affected. Genetic modifications of stem cells have great therapeutic potentials and our results picture a dynamic embryonic genome response to ectopic transgene integration that may have important implications in the future safety and efficacy modifications of stem cells.
Sugar-Binding Proteins Potently Inhibit Dendritic Cell Human Immunodeficiency Virus Type 1 (HIV-1) Infection and Dendritic-Cell-Directed HIV-1 Transfer
Authors: Turville Stuart G. , Vermeire Kurt , Balzarini Jan , Schols Dominique
Published: October 2005

Both endocytic uptake and viral fusion can lead to human immunodeficiency virus type 1 (HIV-1) transfer to CD4
+
lymphocytes, either through directional regurgitation (infectious transfer in
trans
[I-IT]) or through de novo viral production in dendritic cells (DCs) resulting in a second-phase transfer to CD4
+
lymphocytes (infectious second-phase transfer [I-SPT]). We have evaluated in immature monocyte-derived DCs both pathways of transfer with regard to their susceptibilities to being blocked by potential microbicidal compounds, including cyanovirin (CNV); the plant lectins
Hippeastrum hybrid
agglutinin,
Galanthus nivalis
agglutinin,
Urtica dioica
agglutinin, and
Cymbidium hybrid
agglutinin; and the glycan mannan. I-IT was a relatively inefficient means of viral transfer compared to I-SPT at both high and low levels of the viral inoculum. CNV was able to completely block I-IT at 15 μg/ml. All other compounds except mannan could inhibit I-IT by at least 90% when used at doses of 15 μg/ml. In contrast, efficient inhibition of I-SPT was remarkably harder to achieve, as 50% effective concentration levels for plant lectins and CNV to suppress this mode of HIV-1 transfer increased significantly. Thus, our findings indicate that I-SPT may be more elusive to targeting by antiviral drugs and stress the need for drugs affecting the pronounced inhibition of the infection of DCs by HIV-1.
The Herpesvirus Saimiri Replication and Transcription Activator Acts Synergistically with CCAAT Enhancer Binding Protein Alpha To Activate the DNA Polymerase Promoter
Authors: Wakenshaw Louise , Walters Matthew S. , Whitehouse Adrian
Published: October 2005

The open reading frame (ORF) 50 gene product, also known as the replication and transcription activator (Rta), is an immediate-early gene which is well conserved among all gamma-2 herpesviruses and plays a pivotal role in regulating the latent-lytic switch. Herpesvirus saimiri (HVS) ORF 50a functions as a sequence-specific transactivator capable of activating delayed-early (DE) gene expression via binding directly to an ORF 50 response element (RE) within the respective promoter. Analysis of the ORF 50 REs have identified two distinct types within HVS gene promoters. The first comprises a consensus sequence motif, CCN
9
GG, the second an AT-rich sequence. Here we demonstrate that ORF 50a is capable of transactivating the DE ORF 9 promoter which encodes the DNA polymerase. Deletion analysis of the ORF 9 promoter mapped the ORF 50 RE to a 95-bp region situated 126 bp upstream of the initiation codon. Gel retardation analysis further mapped the RE to a 28-bp fragment, which was able to confer ORF 50 responsiveness on an enhancerless simian virus 40 minimal promoter. Furthermore, sequence analysis identified multiple CCAAT enhancer binding protein alpha (C/EBPα) binding sites within the ORF 9 promoter and specifically two within the close vicinity of the AT-rich ORF 50 RE. Analysis demonstrated that the HVS ORF 50a and C/EBPα proteins associate with the ORF 9 promoter in vivo, interact directly, and synergistically activate the ORF 9 promoter by binding to adjacent binding motifs. Overall, these data suggest a cooperative interaction between HVS ORF 50a and C/EBPα proteins to activate the DNA polymerase promoter during early stages of the lytic replication cycle.
How Many Human Immunodeficiency Virus Type 1-Infected Target Cells Can a Cytotoxic T-Lymphocyte Kill?
Authors: Wick W. David , Yang Otto O. , Corey Lawrence , Self Steven G.
Published: October 2005

The antiviral role of CD8+cytotoxic T lymphocytes (CTLs) in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. Specifically, the degree to which CTLs reduce viral replication by killing HIV-1-infected cells in vivo is not known. Here we employ mathematical models of the infection process and CTL action to estimate the rate that CTLs can kill HIV-1-infected cells from in vitro and in vivo data. Our estimates, which are surprisingly consistent considering the disparities between the two experimental systems, demonstrate that on average CTLs can kill from 0.7 to 3 infected target cells per day, with the variability in this figure due to epitope specificity or other factors. These results are compatible with the observed decline in viremia after primary infection being primarily a consequence of CTL activity and have interesting implications for vaccine design.
Quantitative Analysis of the Hepatitis C Virus Replication Complex
Authors: Quinkert Doris , Bartenschlager Ralf , Lohmann Volker
Published: October 2005

The hepatitis C virus (HCV) encodes a large polyprotein; therefore, all viral proteins are produced in equimolar amounts regardless of their function. The aim of our study was to determine the ratio of nonstructural proteins to RNA that is required for HCV RNA replication. We analyzed Huh-7 cells harboring full-length HCV genomes or subgenomic replicons and found in all cases a >1,000-fold excess of HCV proteins over positive- and negative-strand RNA. To examine whether all nonstructural protein copies are involved in RNA synthesis, we isolated active HCV replication complexes from replicon cells and examined them for their content of viral RNA and proteins before and after treatment with protease and/or nuclease. In vitro replicase activity, as well as almost the entire negative- and positive-strand RNA, was resistant to nuclease treatment, whereas <5% of the nonstructural proteins were protected from protease digest but accounted for the full in vitro replicase activity. In consequence, only a minor fraction of the HCV nonstructural proteins was actively involved in RNA synthesis at a given time point but, due to the high amounts present in replicon cells, still representing a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, we estimate that an active HCV replicase complex consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred nonstructural protein copies, which might be required as structural components of the vesicular compartments that are the site of HCV replication.
Mechanism for Removal of Tumor Necrosis Factor Receptor 1 from the Cell Surface by the Adenovirus RIDα/β Complex
Authors: Chin Y. Rebecca , Horwitz Marshall S.
Published: October 2005

Proteins encoded in adenovirus early region 3 have important immunoregulatory properties. We have recently shown that the E3-10.4K/14.5K (RIDα/β) complex downregulates tumor necrosis factor receptor 1 (TNFR1) expression at the plasma membrane. To study the role of the RIDβ tyrosine sorting motif in the removal of surface TNFR1, tyrosine 122 on RIDβ was mutated to alanine or phenylalanine. Both RIDβ mutations not only abolished the downregulation of surface TNFR1 but paradoxically increased surface TNFR1 levels. RID also downregulates other death receptors, such as FAS; however, surface FAS expression was not increased by RIDβ mutants, suggesting that regulation of TNFR1 and that of FAS by RID are mechanistically different. In the mixing experiments, the wild-type (WT) RID-mediated TNFR1 downregulation was partially inhibited in the presence of RIDβ mutants, indicating that the mutants compete for TNFR1 access. Indeed, an association between RIDβ and TNFR1 was shown by coimmunoprecipitation. In contrast, the mutants did not affect the WT RID-induced downregulation of FAS. These differential effects support a model in which RID associates with TNFR1 on the plasma membrane, whereas RID probably associates with FAS in a cytoplasmic compartment. By using small interfering RNA against the μ2 subunit of adaptor protein 2, dominant negative dynamin construct K44A, and the lysosomotropic agents bafilomycin A1 and ammonium chloride, we also demonstrated that surface TNFR1 was internalized by RID by a clathrin-dependent process involving μ2 and dynamin, followed by degradation of TNFR1 via an endosomal/lysosomal pathway.
Selection of Muscle-Binding Peptides from Context-Specific Peptide-Presenting Phage Libraries for Adenoviral Vector Targeting
Authors: Ghosh Debadyuti , Barry Michael A.
Published: October 2005

Production of cell-targeting vectors in part involves the addition of new targeting ligands to the vector to mediate binding to the cells of interest. For viral vectors, the ideal approach is to genetically engineer new ligands into the capsid proteins of the virus to generate a single agent to mediate therapy. Although this is ideal, this insertion of an exogenous ligand from one structural context into the differing structural context of a capsid protein can ablate the function of the ligand or disrupt viral assembly and function. To address this context problem for adenoviral vectors, we have engineered a “context-specific” peptide-presenting phage library. We have displayed a 12-amino-acid (12-mer) random peptide library between the H and I sheets of the fiber protein of adenovirus type 5 on the pIII protein of fd bacteriophage. This library was used for peptide selection against C2C12 mouse skeletal muscle cells. Five rounds of selection combined with four rounds of clearing on nontarget cells selected one primary peptide designated 12.51, which bound target C2C12 cells approximately 100-fold better than the positive control RGD peptide. Translation of 12.51 back into the fiber protein produced a ligand-modified adenoviral vector that mediated 14-fold-better transduction of target C2C12 cells. These data suggest context-specific peptide-presenting libraries may allow selection of compatible peptide ligands for functional translation into viral vectors for retargeting.
Influenza Virus Hemagglutinin (H3 Subtype) Requires Palmitoylation of Its Cytoplasmic Tail for Assembly: M1 Proteins of Two Subtypes Differ in Their Ability To Support Assembly
Authors: Chen Benjamin J. , Takeda Makoto , Lamb Robert A.
Published: October 2005

The influenza A virus hemagglutinin (HA) transmembrane domain boundary region and the cytoplasmic tail contain three cysteines (residues 555, 562, and 565 for the H3 HA subtype) that are highly conserved among the 16 HA subtypes and which are each modified by the covalent addition of palmitic acid. Previous analysis of the role of these conserved cysteine residues led to differing data, suggesting either no role for HA palmitoylation or an important role for HA palmitoylation. To reexamine the role of these residues in the influenza virus life cycle, a series of cysteine-to-serine mutations were introduced into the HA gene of influenza virus A/Udorn/72 (Ud) (H3N2) by using a highly efficient reverse genetics system. Mutant viruses containing HA-C562S and HA-C565S mutations had reduced growth and failed to form plaques in MDCK cells but formed wild-type-like plaques in an MDCK cell line expressing wild-type HA. In cell-cell fusion assays, nonpalmitoylated H3 HA, in both cDNA-transfected and virus-infected cells, was fully competent for HA-mediated membrane fusion. When the HA cytoplasmic tail cysteine mutants were examined for lipid raft association, using as the criterion Triton X-100 insolubility, loss of raft association did not show a direct correlation with a reduction in virus replication. However, mutant virus assembly was reduced in parallel with reduced virus replication. Additionally, a reassortant of strain A/WSN/33 (WSN), containing the Ud HA gene with mutations C555S, C562S, and C565S, produced virus that could form plaques on regular MDCK cells and had only moderately decreased replication, suggesting differences in the interactions between Ud and WSN HA and internal viral proteins. Analysis of M1 mutants containing substitutions in the six residues that differ between the Ud and WSN M1 proteins indicated that a constellation of residues are responsible for the difference between the M1 proteins in their ability to support virus assembly with nonpalmitoylated H3 HA.
Alternate Polypurine Tracts (PPTs) Affect the Rous Sarcoma Virus RNase H Cleavage Specificity and Reveal a Preferential Cleavage following a GA Dinucleotide Sequence at the PPT-U3 Junction
Authors: Chang Kevin W. , Julias John G. , Alvord W. Gregory , Oh Jangsuk , Hughes Stephen H.
Published: October 2005

Retroviral polypurine tracts (PPTs) serve as primers for plus-strand DNA synthesis during reverse transcription. The generation and removal of the PPT primer requires specific cleavages by the RNase H activity of reverse transcriptases; removal of the PPT primer defines the left end of the linear viral DNA. We replaced the endogenous PPT from RSVP(A)Z, a replication-competent shuttle vector based on Rous sarcoma virus (RSV), with alternate retroviral PPTs and the duck hepatitis B virus “PPT.” Viruses in which the endogenous RSV PPT was replaced with alternate PPTs had lower relative titers than the wild-type virus. 2-LTR circle junction analysis showed that the alternate PPTs caused significant decreases in the fraction of viral DNAs with complete (consensus) ends and significant increases in the insertion of part or all of the PPT at the 2-LTR circle junctions. The last two nucleotides in the 3′ end of the RSV PPT are GA. Examination of the (mis)cleavages of the alternate PPTs revealed preferential cleavages after GA dinucleotide sequences. Replacement of the terminal 3′ A of the RSV PPT with G caused a preferential miscleavage at a GA sequence spanning the PPT-U3 boundary, resulting in the deletion of the terminal adenine normally present at the 5′ end of the U3. A reciprocal G-to-A substitution at the 3′ end of the murine leukemia virus PPT increased the relative titer of the chimeric RSV-based virus and the fraction of consensus 2-LTR circle junctions.
Role of N-Linked Glycans on Bunyamwera Virus Glycoproteins in Intracellular Trafficking, Protein Folding, and Virus Infectivity
Authors: Shi Xiaohong , Brauburger Kristina , Elliott Richard M.
Published: October 2005

The membrane glycoproteins (Gn and Gc) of
Bunyamwera virus
(BUN, family
Bunyaviridae
) contain three potential sites for the attachment of N-linked glycans: one site (N60) on Gn and two (N624 and N1169) on Gc. We determined that all three sites are glycosylated. Digestion of the glycoproteins with endo-β-
N
-acetylglucosaminidase H (endo H) or peptide:
N
-glycosidase F revealed that Gn and Gc differ significantly in their glycan status and that late in infection Gc glycans remain endo H sensitive. The roles of the N-glycans in intracellular trafficking of the glycoproteins to the Golgi, protein folding, and virus replication were investigated by mutational analysis and confocal immunofluorescence. Elimination of the glycan on Gn, by changing N60 to a Q residue, resulted in the protein misfolding and failure of both Gn and Gc proteins to traffic to the Golgi complex. We were unable to rescue a viable virus by reverse genetics from a cDNA containing the N60Q mutation. In contrast, mutant Gc proteins lacking glycans on either N624 or N1169, or both sites, were able to target to the Golgi. Gc proteins containing mutations N624Q and N1169Q acquired endo H resistance. Three viable N glycosylation-site-deficient viruses, lacking glycans on one site or both sites on Gc, were created by reverse genetics. The viability of these recombinant viruses and analysis of growth kinetics indicates that the glycans on Gc are not essential for BUN replication, but they do contribute to the efficiency of virus infection.
Distinct Chemokine Triggers and In Vivo Migratory Paths of Fluorescein Dye-Labeled T Lymphocytes in Acutely Simian Immunodeficiency Virus SIV
mac251
-Infected and Uninfected Macaques
Authors: Clay Candice C. , Rodrigues Denise S. , Harvey Danielle J. , Leutenegger Christian M. , Esser Ursula
Published: October 2005

To define the possible impact of T-lymphocyte trafficking parameters on simian immunodeficiency virus (SIV) pathogenesis, we examined migratory profiles of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T lymphocytes in acutely SIV
mac251
-infected and uninfected macaques within 48 h after autologous transfer. Despite significant upregulation of homeostatic chemokine CCL19/macrophage inflammatory protein 3β and proinflammatory chemokine CXCL9/monokine induced by gamma interferon in secondary lymphoid tissue in SIV infection, no differences in CFSE
+
T-lymphocyte frequencies or cell compartmentalization in lymph nodes were identified between animal groups. By contrast, a higher frequency of CFSE
+
T lymphocytes in the small intestine was detected in acute SIV infection. This result correlated with increased numbers of gut CD4 T lymphocytes expressing chemokine receptors CCR9, CCR7, and CXCR3 and high levels of their respective chemokine ligands in the small intestine. The changes in trafficking parameters in SIV-infected macaques occurred concomitantly with acute gut CD4 T-lymphocyte depletion. Here, we present the first in vivo T-lymphocyte trafficking study in SIV infection and a novel approach to delineate T-lymphocyte recruitment into tissues in the nonhuman primate animal model for AIDS. Such studies are likely to provide unique insights into T-lymphocyte sequestration in distinct tissue compartments and possible mechanisms of CD4 T-lymphocyte depletion and immune dysfunction in simian AIDS.
Dissecting the Interferon-Induced Inhibition of Hepatitis C Virus Replication by Using a Novel Host Cell Line
Authors: Windisch Marc P. , Frese Michael , Kaul Artur , Trippler Martin , Lohmann Volker , Bartenschlager Ralf
Published: October 2005

The
Hepatitis C virus
(HCV), a member of the family
Flaviviridae
, is a major cause of chronic liver disease.
Patients are currently treated with alpha interferon (IFN-α)
that is given alone or in combination with ribavirin. Unfortunately,
this treatment is ineffective in eliminating the virus in a large
proportion of individuals. IFN-induced antiviral activities have been
intensively studied in the HCV replicon system. It was found that both
IFN-α and IFN-γ inhibit HCV replicons, but the
underlying mechanisms have not yet been identified. Of note is that
nearly all of these studies were performed with the human hepatoma cell
line Huh-7. Here, we report that genotypes 1b and 2a replicons also
replicate in the human hepatoblastoma cell line HuH6. Similar to what
has been described for Huh-7 cells, we observed that efficient HCV
replication in HuH6 cells depends on the presence of cell
culture-adaptive mutations and the permissiveness of the host cell.
However, three major differences exist: in HuH6 cells, viral
replication is (i) independent from ongoing cell proliferation, (ii)
less sensitive to certain antiviral compounds, and (iii) highly
resistant to IFN-γ. The latter is not due to a general defect
in IFN signaling, as IFN-γ induces the nuclear translocation of
signal transducer and activator of transcription 1 (STAT1), the
enhanced transcription of several IFN-regulated genes, and the
inhibition of unrelated viruses such as influenza A virus and Semliki
Forest virus. Taken together, the results establish HuH6 replicon cells
as a valuable tool for IFN studies and for the evaluation of antiviral
compounds.
Severe Acute Respiratory Syndrome Coronavirus Fails To Activate Cytokine-Mediated Innate Immune Responses in Cultured Human Monocyte-Derived Dendritic Cells
Authors: Ziegler Thedi , Matikainen Sampsa , Rönkkö Esa , Österlund Pamela , Sillanpää Maarit , Sirén Jukka , Fagerlund Riku , Immonen Milla , Melén Krister , Julkunen Ilkka
Published: October 2005

Activation of host innate immune responses was studied in severe acute respiratory syndrome coronavirus (SCV)-infected human A549 lung epithelial cells, macrophages, and dendritic cells (DCs). In all cell types, SCV-specific subgenomic mRNAs were seen, whereas no expression of SCV proteins was found. No induction of cytokine genes (alpha interferon [IFN-α], IFN-β, interleukin-28A/B [IL-28A/B], IL-29, tumor necrosis factor alpha, CCL5, or CXCL10) or IFN-α/β-induced MxA gene was seen in SCV-infected A549 cells, macrophages, or DCs. SCV also failed to induce DC maturation (CD86 expression) or enhance major histocompatibility complex class II expression. Our data strongly suggest that SCV fails to activate host cell cytokine gene expression in human macrophages and DCs.
Cloning of the Chicken RNA Polymerase I Promoter and Use for Reverse Genetics of Influenza A Viruses in Avian Cells
Authors: Massin Pascale , Rodrigues Pierre , Marasescu Monica , van der Werf Sylvie , Naffakh Nadia
Published: October 2005

Reverse genetics techniques to rescue influenza viruses have thus far been based on the use of a human polymerase I (PolI) promoter to direct the synthesis of the eight viral RNAs. They can only be used on cells from primate origin due to the species specificity of the PolI promoter. Here we report the cloning of the chicken PolI promoter sequence and the generation of recombinant influenza virus upon transfection of bidirectional PolI/PolII plasmids in avian cells. Potential contributions of this new reverse genetics system in the fields of influenza virus research and influenza vaccine production are discussed.
Mutation of a Single Amino Acid Residue in the Basic Region of the Epstein-Barr Virus (EBV) Lytic Cycle Switch Protein Zta (BZLF1) Prevents Reactivation of EBV from Latency
Authors: Schelcher Celine , Valencia Sarah , Delecluse Henri-Jacques , Hicks Matthew , Sinclair Alison J.
Published: October 2005

Zta, the product of the
BZLF1
gene carried by Epstein-Barr virus (EBV), is crucial for reactivation of EBV from latency. Zta is a member of the bZIP family of transcription factors, and in common with many of these, Zta possesses a conserved cysteine residue in its basic region (C189) and a further cysteine residue in its ZIP region (C222). We demonstrate that C189 is required to reactivate EBV from latency but C222 is not and that this single amino acid affects two independent functions of Zta, (i) binding to a Zta-responsive site and (ii) manipulating the cell cycle.
3D seismic analysis of circular evaporite dissolution structures, Eastern Mediterranean
Authors: Bertoni C. , Cartwright J.A.
Published: October 2005

Buried circular collapse structures above a tabular evaporitic body are recorded by recently acquired 3D seismic data on the Levant Basin and continental margin, offshore Israel (Eastern Mediterranean). The structures formed during the Pliocene as buried Messinian (late Miocene) evaporites underwent extensive dissolution in a submarine, deep-water setting. Three-dimensional seismic analysis is used to describe the detailed morphology of the structures and the associated overburden, allowing the reconstruction of their origin and development. It is proposed that evaporite dissolution led to the collapse of the weakly lithified overburden, and this deformed with a series of concentric extensional faults. From the structural analysis of the overburden, the estimated maximum duration of the dissolution event is 0.75–1 Ma. The mechanism proposed for the creation of the circular collapse structures is subjacent dissolution of the more soluble evaporites in the Messinian evaporites, as a result of focused vertical fluid flow at the base of the evaporitic series. Rapid release of overpressured fluids, as, for example, during an earthquake, is thought to have initiated the focused fluid flow, which impinged on the evaporitic seal to the point where dissolution occurred, creating the localized circular collapse structures in the overburden.