bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human Cytomegalovirus pp28 (UL99) Localizes to a Cytoplasmic Compartment Which Overlaps the Endoplasmic Reticulum-Golgi-Intermediate Compartment
Authors: Sanchez Veronica , Sztul Elizabeth , Britt William J. 
Published: July 2002

ABSTRACT

Although the assembly of herpesviruses has remained an active area of investigation, considerable controversy continues to surround the cellular location of tegument and envelope acquisition. This controversy is particularly evident when the proposed pathways for α- and β-herpesvirus assembly are compared. We have approached this aspect of human cytomegalovirus (HCMV) assembly, specifically, envelopment, by investigating the intracellular trafficking of viral tegument proteins which localize in the cytoplasms of infected cells. In this study we have demonstrated that the virion tegument protein pp28 (UL99), a true late protein, was membrane associated as a result of myristoylation. A mutation in this protein which prevented incorporation of [
3
H]myristic acid also altered the detergent solubility and intracellular distribution of the protein when it was expressed in transfected cells. Using a panel of markers for intracellular compartments, we could localize the expression of wild-type pp28 to an intracellular compartment which colocalized with the endoplasmic reticulum-Golgi-intermediate compartment (ERGIC), a dynamic compartment of the secretory pathway which interfaces with both the ER and Golgi apparatus. The localization of this viral tegument protein within an early secretory compartment of the cell provided further evidence that the assembly of the HCMV tegument likely includes a cytoplasmic phase. Because pp28 has been shown to be localized to a cytoplasmic assembly compartment in HCMV-infected cells, our findings also suggested that viral tegument protein interactions within the secretory pathway may have an important role in the assembly of the virion.
A Single Amino Acid Change in nsP1 Attenuates Neurovirulence of the Sindbis-Group Alphavirus S.A.AR86
Authors: Heise Mark T. , Simpson Dennis A. , Johnston Robert E. 
Published: July 2002

ABSTRACTS.A.AR86, a member of the Sindbis group of alphaviruses, is neurovirulent in adult mice and has a unique threonine at position 538 of nsP1; nonneurovirulent members of this group of alphaviruses encode isoleucine. Isoleucine was introduced at position 538 in the wild-type S.A.AR86 infectious clone, ps55, and virus derived from this mutant clone, ps51, was significantly attenuated for neurovirulence compared to that derived from ps55. Intracranial (i.c.) s55 infection resulted in severe disease, including hind limb paresis, conjunctivitis, weight loss, and death in 89% of animals. In contrast, s51 caused fewer clinical signs and no mortality. Nevertheless, comparison of the virus derived from the mutant (ps51) and wild-type (ps55) S.A.AR86 molecular clones demonstrated that s51 grew as well as or better than the wild-type s55 virus in tissue culture and that viral titers in the brain following i.c. infection with s51 were equivalent to those of wild-type s55 virus. Analysis of viral replication within the brain by in situ hybridization revealed that both viruses established infection in similar regions of the brain at early times postinfection (12 to 72 h). However, at late times postinfection, the wild-type s55 virus had spread throughout large areas of the brain, while the s51 mutant exhibited a restricted pattern of replication. This suggests that s51 is either defective in spreading throughout the brain at late times postinfection or is cleared more rapidly than s55. Further evidence for the contribution of nsP1 Thr 538 to S.A.AR86 neurovirulence was provided by experiments in which a threonine residue was introduced at nsP1 position 538 of Sindbis virus strain TR339, which is nonneurovirulent in weanling mice. The resulting virus, 39ns1, demonstrated significantly increased neurovirulence and morbidity, including weight loss and hind limb paresis. These results demonstrate a role for alphavirus nonstructural protein genes in adult mouse neurovirulence.
Evaluation of Cytotoxic T-Lymphocyte Responses in Human and Nonhuman Primate Subjects Infected with Human Immunodeficiency Virus Type 1 or Simian/Human Immunodeficiency Virus
Authors: Fu Tong-Ming , Freed Daniel C. , Trigona Wendy L. , Guan Liming , Zhu Lan , Long Romnie , Persaud Natasha V. , Manson Kelledy , Dubey Sheri , Shiver John W. 
Published: July 2002

ABSTRACTCytotoxic T-lymphocyte (CTL) responses have been implicated as playing an important role in control of human immunodeficiency virus (HIV) infection. However, it is technically difficult to demonstrate CTL responses consistently in nonhuman primate and human subjects using traditional cytotoxicity assay methods. In this study, we systematically evaluated culture conditions that may affect the proliferation and expansion of CTL effector cells and presented a sensitive method for detection of cytotoxicity responses with bulk CTL cultures. We confirmed the sensitivity and specificity of this method by demonstration of vigorous CTL responses in a simian-HIV (SHIV)-infected rhesus macaque. The expansion of epitope-specific CTL effector cells was also measured quantitatively by CTL epitope-major histocompatibility complex tetramer complex staining. In addition, two new T-cell determinants in the SIVgagregion are identified. Last, we showed the utility of this method for studying CTL responses in chimpanzee and human subjects.
“Rule of Six”: How Does the Sendai Virus RNA Polymerase Keep Count?
Authors: Vulliémoz Diane , Roux Laurent 
Published: July 2002

ABSTRACT

The “rule of six” stipulates that the
Paramyxovirus
RNA polymerase efficiently replicates only viral genomes counting 6
n
+ 0 nucleotides. Because the nucleocapsid proteins (N) interact with 6 nucleotides, an exact nucleotide-N match at the RNA 3′-OH end (3′-OH congruence) may be required for recognition of an active replication promoter. Alternatively, assuming that the six positions for the interaction of N with the nucleotides are not equivalent, the nucleotide position relative to N may be critical (N phase context). The replication abilities of various minireplicons, designed so that the 3′-OH congruence could be discriminated from the N phase context, were studied. The results strongly suggest that the application of the rule of six depends on the recognition of nucleotides positioned in the proper N phase context.
Human Papillomavirus Virus-Like Particles Are Efficient Oral Immunogens when Coadministered with
Escherichia coli
Heat-Labile Enterotoxin Mutant R192G or CpG DNA
Authors: Gerber S. , Lane C. , Brown D. M. , Lord E. , DiLorenzo M. , Clements J. D. , Rybicki E. , Williamson A.-L. , Rose R. C. 
Published: July 2002

ABSTRACT

Certain human papillomaviruses (HPVs) cause most cervical cancer, which remains a significant source of morbidity and mortality among women worldwide. HPV recombinant virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease and are now being evaluated as a parenteral vaccine modality in human subjects. Vaccines formulated for injection generally are more costly, more difficult to administer, and less acceptable to recipients than are mucosally administered vaccines. Since oral delivery represents an attractive alternative to parenteral injection for large-scale human vaccination, the oral immunogenicity of HPV type 11 (HPV-11) VLPs in mice was previously investigated; it was found that a modest systemic neutralizing antibody response was induced (R. C. Rose, C. Lane, S. Wilson, J. A. Suzich, E. Rybicki, and A. L. Williamson, Vaccine 17:2129–2135, 1999). Here we examine whether VLPs of other genotypes may also be immunogenic when administered orally and whether mucosal adjuvants can be used to enhance VLP oral immunogenicity. We show that HPV-16 and HPV-18 VLPs are immunogenic when administered orally and that oral coadministration of these antigens with
Escherichia coli
heat-labile enterotoxin (LT) mutant R192G (LT R192G) or CpG DNA can significantly improve anti-VLP humoral responses in peripheral blood and in genital mucosal secretions. Our results also suggest that LT R192G may be superior to CpG DNA in this ability. These findings support the concept of oral immunization against anogenital HPV disease and suggest that clinical studies involving this approach may be warranted.
Molecular Basis for Cell Tropism of CXCR4-Dependent Human Immunodeficiency Virus Type 1 Isolates
Authors: Tokunaga Kenzo , Greenberg Michael L. , Morse Michael A. , Cumming R. Ian , Lyerly H. Kim , Cullen Bryan R. 
Published: July 2002

ABSTRACTLaboratory isolates of human immunodeficiency virus type 1 (HIV-1) that utilize CXCR4 as a coreceptor infect primary human macrophages inefficiently even though these express a low but detectable level of cell surface CXCR4. In contrast, infection of primary macrophages by primary CXCR4-tropic HIV-1 isolates is readily detectable. Here, we provide evidence suggesting that this difference in cell tropism results from a higher requirement for cell surface CXCR4 for infection by laboratory HIV-1 isolates. Transfected COS7 cells that express a high level of CD4 but a low level of CXCR4 were infected significantly more efficiently by two primary CXCR4-tropic HIV-1 isolates compared to the prototypic laboratory HIV-1 isolate IIIB. More importantly, overexpression of either wild-type or signaling-defective CXCR4 on primary macrophages dramatically enhanced the efficiency of infection by the laboratory HIV-1 isolate yet only modestly enhanced infection by either primary CXCR4-tropic virus. Overexpression of CD4 had, in contrast, only a limited effect on macrophage infection by the laboratory HIV-1, although infection by the primary isolates was markedly enhanced. We therefore conclude that the laboratory CXCR4-tropic HIV-1 isolate exhibits a significantly higher CXCR4 requirement for efficient infection than do the primary CXCR4-tropic isolates and that this difference can explain the poor ability of the laboratory HIV-1 isolate to replicate in primary macrophages. More generally, we propose that the cell tropisms displayed by different strains of HIV-1 in culture can largely be explained on the basis of differential requirements for cell surface CD4 and/or coreceptor expression levels.
Phage Display of Adenovirus Type 5 Fiber Knob as a Tool for Specific Ligand Selection and Validation
Authors: Pereboev Alexander , Pereboeva Larisa , Curiel David T. 
Published: July 2002

ABSTRACT
Adenovirus (Ad) vectors are most potent for use as gene delivery vehicles to infect human cells in vitro and in vivo with high efficiency. The main limitation in utilization of Ad as a gene transfer vector is the lack of specificity. Genetic modifications of Ad capsid proteins resulting in incorporation of foreign polypeptide ligand sequences can redirect the vector towards target cells. However, in many cases the incorporated ligands lose specificity or lead to conformational changes influencing virion integrity. In order to select target-specific ligands a priori structurally compatible with Ad, we propose a system for displaying polypeptide sequences in the context of the Ad fiber knob on the surfaces of filamentous bacteriophages. To establish this concept, we displayed the wild-type Ad serotype 5 knob and knobs containing c-Myc epitopes and six-histidine sequences in the pJuFo phage system. The knobs remained trimeric and bound the coxsackievirus-Ad receptor, and the phage knob-displayed ligands recognized and bound their cognates in the phage-displayed knob context. Further development of this system may be useful for candidate ligand fidelity and Ad structural compatibility validation prior to Ad modification.
Functional Cooperation of Epstein-Barr Virus Nuclear Antigen 2 and the Survival Motor Neuron Protein in Transactivation of the Viral LMP1 Promoter
Authors: Voss Marc D. , Hille Annette , Barth Stephanie , Spurk Andreas , Hennrich Frank , Holzer Daniela , Mueller-Lantzsch Nikolaus , Kremmer Elisabeth , Grässer Friedrich A. 
Published: July 2002

ABSTRACT
Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for viral transformation of B cells and transactivates cellular and viral target genes by binding RBPJκ tethered to cognate promoter elements. EBNA2 interacts with the DEAD-box protein DP103 (DDX20/Gemin3), which in turn is complexed to the survival motor neuron (SMN) protein. SMN is implicated in RNA processing, but a role in transcriptional regulation has also been suggested. Here, we show that DP103 and SMN are complexed in B cells and that SMN coactivates the viral LMP promoter in the presence of EBNA2 in reporter gene assays and in vivo. Subcellular localization studies revealed that nuclear gems and/or coiled bodies containing DP103 and SMN are targeted by EBNA2. Protein-protein interaction experiments demonstrated that DP103 binds to SMN exon 6 and that both EBNA2 and SMN interact with the C terminus of DP103. Furthermore, a DP103 binding-deficient SMN mutant was released from nuclear gems and/or coiled bodies and further enhanced coactivation. In addition, impaired transactivation of a DP103 binding-deficient EBNA2 mutant was rescued by overexpression of SMN. Testing different promoter constructs in luciferase assays showed that RBPJκ is required but not sufficient for coactivation by EBNA2 and SMN. Overall, our data suggest that EBNA2 might target spliceosomal complexes by binding to DP103, thereby releasing SMN which subsequently exerts a coactivational function within the RNA-polymerase II transcription complex on the LMP1 promoter.
Antiapoptotic Function of Cdk9 (TAK/P-TEFb) in U937 Promonocytic Cells
Authors: Foskett Shannon M. , Ghose Romi , Tang Derek Ng , Lewis Dorothy E. , Rice Andrew P. 
Published: July 2002

ABSTRACTCdk9 is the catalytic subunit of TAK (cyclinT1/P-TEFb), a cellular protein kinase that mediates human immunodeficiency virus type 1 (HIV-1) Tat transcriptional activation function. To examine Cdk9 function in cells relevant to HIV-1 infection, we used a murine leukemia virus retrovirus vector to transduce and overexpress the cDNA of a dominant negative mutant Cdk9 protein (Cdk9-dn) in Jurkat T cells and U937 promonocytic cells. In Jurkat cells, overexpression of Cdk9-dn specifically inhibited Tat transactivation and HIV-1 replication but had no inhibitory effect on induction of CD69, CD25, and interleukin-2 following T-cell activation. In U937 cells, overexpression of Cdk9-dn sensitized cells to apoptosis, especially after phorbol myristate acetate (PMA) treatment to induce differentiation to macrophage-like cells. Because Cdk9 function is induced in PMA-treated U937 cells, Cdk9 may play an antiapoptotic role during monocyte differentiation.
Does a cdc2 Kinase-Like Recognition Motif on the Core Protein of Hepadnaviruses Regulate Assembly and Disintegration of Capsids?
Authors: Barrasa M. Inmaculada , Guo Ju-Tao , Saputelli Jeffrey , Mason William S. , Seeger Christoph 
Published: July 2002

ABSTRACT
Hepadnaviruses are enveloped viruses, each with a DNA genome packaged in an icosahedral nucleocapsid, which is the site of viral DNA synthesis. In the presence of envelope proteins, DNA-containing nucleocapsids are assembled into virions and secreted, but in the absence of these proteins, nucleocapsids deliver viral DNA into the cell nucleus. Presumably, this step is identical to the delivery of viral DNA during the initiation of an infection. Unfortunately, the mechanisms triggering the disintegration of subviral core particles and delivery of viral DNA into the nucleus are not yet understood. We now report the identification of a sequence motif resembling a serine- or threonine-proline kinase recognition site in the core protein at a location that is required for the assembly of core polypeptides into capsids. Using duck hepatitis B virus, we demonstrated that mutations at this sequence motif can have profound consequences for RNA packaging, DNA replication, and core protein stability. Furthermore, we found a mutant with a conditional phenotype that depended on the cell type used for virus replication. Our results support the hypothesis predicting that this motif plays a role in assembly and disassembly of viral capsids.
Loss of a Single N-Linked Glycan Allows CD4-Independent Human Immunodeficiency Virus Type 1 Infection by Altering the Position of the gp120 V1/V2 Variable Loops
Authors: Kolchinsky Peter , Kiprilov Enko , Bartley Peter , Rubinstein Roee , Sodroski Joseph 
Published: July 2002

ABSTRACTThe gp120 envelope glycoprotein of primary human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and the CCR5 chemokine receptor on the target cell. Previously, we adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for CD4-independent replication were limited to the V2 loop-V1/V2 stem. Here we show that elimination of a single glycosylation site at asparagine 197 in the V1/V2 stem is sufficient for CD4-independent gp120 binding to CCR5 and for HIV-1 entry into CD4-negative cells expressing CCR5. Deletion of the V1/V2 loops also allowed CD4-independent viral entry and gp120 binding to CCR5. The binding of the wild-type ADA gp120 to CCR5 was less dependent upon CD4 at 4°C than at 37°C. In the absence of the V1/V2 loops, neither removal of the N-linked carbohydrate at asparagine 197 nor lowering of the temperature increased the CD4-independent phenotypes. A CCR5-binding conformation of gp120, achieved by CD4 interaction or by modification of temperature, glycosylation, or variable loops, was preferentially recognized by the monoclonal antibody 48d. These results suggest that the CCR5-binding region of gp120 is occluded by the V1/V2 variable loops, the position of which can be modulated by temperature, CD4 binding, or an N-linked glycan in the V1/V2 stem.
Genetic Evidence for a Structural Interaction between the Carboxy Termini of the Membrane and Nucleocapsid Proteins of Mouse Hepatitis Virus
Authors: Kuo Lili , Masters Paul S. 
Published: July 2002

ABSTRACT
The coronavirus membrane (M) protein is the most abundant virion protein and the key component in viral assembly and morphogenesis. The M protein of mouse hepatitis virus (MHV) is an integral membrane protein with a short ectodomain, three transmembrane segments, and a large carboxy-terminal endodomain facing the interior of the viral envelope. The carboxy terminus of MHV M has previously been shown to be extremely sensitive to mutation, both in a virus-like particle expression system and in the intact virion. We have constructed a mutant, MΔ2, containing a two-amino-acid truncation of the M protein that was previously thought to be lethal. This mutant was isolated by means of targeted RNA recombination with a powerful host range-based selection allowed by the interspecies chimeric virus fMHV (MHV containing the ectodomain of the feline infectious peritonitis virus S protein). Analysis of multiple second-site revertants of the MΔ2 mutant has revealed changes in regions of both the M protein and the nucleocapsid (N) protein that can compensate for the loss of the last two residues of the M protein. Our data thus provide the first genetic evidence for a structural interaction between the carboxy termini of the M and N proteins of MHV. In addition, this work demonstrates the efficacy of targeted recombination with fMHV for the systematic genetic analysis of coronavirus structural protein interactions.
Increased Production of Interleukin-8 in Primary Human Monocytes and in Human Epithelial and Endothelial Cell Lines after Dengue Virus Challenge
Authors: Bosch Irene , Xhaja Kris , Estevez Luis , Raines Gregory , Melichar Heather , Warke Rajas V. , Fournier Marcia V. , Ennis Francis A. , Rothman Alan L. 
Published: July 2002

ABSTRACTThe more severe form of dengue virus infection, dengue hemorrhagic fever, is characterized by plasma leakage and derangements in hemostasis. As elevated interleukin-8 (IL-8) levels have been observed in sera from patients with more severe disease manifestations, a study was initiated to look at the effect of dengue virus infection in vitro on proinflammatory cytokine secretion and expression. A significant increase in IL-8 levels in the culture supernatant of primary human monocytes infected with dengue 2 virus (D2V) New Guinea C (NGC) was found by enzyme-linked immunosorbent assay. Additionally, by reverse transcriptase PCR, the mRNA was also augmented. Among the proinflammatory cytokines and their mRNAs measured (IL-6, IL-1β, IL-8, and tumor necrosis factor alpha), IL-8 showed the greatest change following D2V infection. Similarly, two cell lines, 293T (a human epithelial cell line) and ECV304 (an endothelial cell line), were permissive to D2V NGC and responded to the infection by increasing the synthesis of IL-8. Nuclear factor kappa B (NF-κB) and nuclear factor IL-6 (NFIL-6) are primary mediators of IL-8 expression. We studied the transcriptional regulation of IL-8 in the ECV304 and 293T cell lines and found that the induction of IL-8 gene expression involved the activation of NF-κB (P= 0.001) and, to a lesser extent, the activation of NFIL-6 in ECV304 cells only. We next observed by the chromatin immunoprecipitation procedure in vivo acetylation of core histones bound to the IL-8 promoter after D2V infection. IL-8 produced by infected monocytes and also IL-8 that may be produced by endothelial or other epithelial cells is associated with the hyperacetylation of histones bound to the IL-8 promoter in addition to the activation of transcription by NF-κB. We hypothesize that the overall increase in IL-8 synthesis observed in this in vitro study may play a role in the pathogenesis of the plasma leakage seen in dengue hemorrhagic fever and dengue shock syndrome.
U
S
3 Protein Kinase of Herpes Simplex Virus 1 Blocks Caspase 3 Activation Induced by the Products of U
S
1.5 and U
L
13 Genes and Modulates Expression of Transduced U
S
1.5 Open Reading Frame in a Cell Type-Specific Manner
Authors: Hagglund Ryan , Munger Joshua , Poon Alice P. W. , Roizman Bernard 
Published: July 2002

ABSTRACT

The coding domain of the herpes simplex virus type 1 (HSV-1) α22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and U
S
1.5, a protein colinear with the carboxyl-terminal domain of ICP22. In HSV-1-infected cells, ICP22 and U
S
1.5 are extensively modified by the U
L
13 and U
S
3 viral protein kinases. In this report, we show that in contrast to other viral proteins defined by their properties as α proteins, U
S
1.5 becomes detectable and accumulated only at late times after infection. Moreover, significantly more U
S
1.5 protein accumulated in cells infected with a mutant lacking the U
L
13 gene than in cells infected with wild-type virus. To define the role of viral protein kinases on the accumulation of U
S
1.5 protein, rabbit skin cells or Vero cells were exposed to recombinant baculoviruses that expressed U
S
1.5, U
L
13, or U
S
3 proteins under a human cytomegalovirus immediate-early promoter. The results were as follows. (i) Accumulation of the U
S
1.5 protein was reduced by concurrent expression of the U
L
13 protein kinase and augmented by concurrent expression of the U
S
3 protein kinase. The magnitude of the reduction or increase in the accumulation of the U
S
1.5 protein was cell type dependent. The effect of U
L
13 kinase appears to be specific inasmuch as it did not affect the accumulation of glycoprotein D in cells doubly infected by recombinant baculoviruses expressing these genes. (ii) The reduction in accumulation of the U
S
1.5 protein was partially due to proteasome-dependent degradation. (iii) Both U
S
1.5 and U
L
13 proteins activated caspase 3, indicative of programmed cell death. (iv) Concurrent expression of the U
S
3 protein kinase blocked activation of caspase 3. The results are concordant with those published elsewhere (J. Munger and B. Roizman, Proc. Natl. Acad. Sci. USA 98:10410–10415, 2001) that the U
S
3 protein kinase can block apoptosis by degradation or posttranslational modification of BAD.