bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Vaccinia Virus E8R Gene Product: a Viral Membrane Protein That Is Made Early in Infection and Packaged into the Virions' Core
Authors: Doglio Laura , De Marco Ario , Schleich Sibylle , Roos Norbert , Krijnse Locker Jacomine 
Published: September 2002

ABSTRACT

Vaccinia virus
(VV), a member of the poxvirus family, is unique among most other DNA viruses in that both transcription and DNA replication occur in the cytoplasm of the host cell. It was recently shown by electron microscopy (EM) that soon after viral DNA synthesis is initiated in HeLa cells, the replication sites become enwrapped by the membrane of the endoplasmic reticulum (ER). In the same study, a novel VV membrane protein, the E8R gene product, that may play a role in the ER wrapping process was identified (N. Tolonen, L. Doglio, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:2031-2046, 2001). In the present study, the gene product of E8R was characterized both biochemically and morphologically. We show that E8R is made predominantly early in infection but is packaged into the virion. On two-dimensional gel electrophoresis, the protein appeared as a single spot throughout the VV life cycle; however, in the assembled virion, the protein underwent several modifications which resulted in a change in its molecular weight and its isoelectric point. EM of labeled cryosections of infected HeLa cells showed that the protein localized to the ER and to membranes located on one side of the Golgi complex as early as 1 h postinfection. Late in infection, E8R was additionally associated with membranes of immature virions and with intracellular mature viruses. Although E8R is predominantly associated with membranes, we show that the protein is associated with viral cores; the protein is present in cores made with NP-40-dithiothreitol as well as in incoming cores, the result of the viral entry process, early in infection. Finally, we show that E8R can be phosphorylated in vitro by the viral kinase F10L. It is able to bind DNA in vitro, and this binding may be modulated by phosphorylation by F10L. A putative role of the E8R gene product throughout the VV life cycle is discussed.
Increased Macrophage Infection upon Subcutaneous Inoculation of Rhesus Macaques with Simian Immunodeficiency Virus-Loaded Dendritic Cells or T Cells but Not with Cell-Free Virus
Authors: Ignatius Ralf , Tenner-Racz Klara , Messmer Davorka , Gettie Agegnehu , Blanchard James , Luckay Amara , Russo Christine , Smith Stephen , Marx Preston A. , Steinman Ralph M. , Racz Paul , Pope Melissa 
Published: September 2002

ABSTRACTInformation on the establishment of immunodeficiency virus infection through transmission of infected cells is sparse. Dendritic cells (DCs) and T cells may be central to the onset and subsequent spread of infection following mucosal exposure. To directly investigate the consequences of virus being introduced by DCs or T cells, we reinjected ex vivo simian immunodeficiency virus (SIV)-loaded autologous immature DCs and T cells subcutaneously (s.c.) into healthy macaques. s.c. injection of cell-bound virus was used to mirror what may happen if virus-loaded cells pass through an epithelium or perhaps DCs and T cells that immediately entrap cell-free virus, having just crossed an epithelial barrier. Virus load in the plasma was monitored along with combined in situ hybridization and immunohistochemistry to identify the cells replicating virus in the lymphoid tissues. Both DCs and T cells transmitted infection after being pulsed with either wild-type ornef-defective (deltanef) SIVmac239. As seen in animals infected intravenously, replication of deltanefwas attenuated compared to that of wild-type virus when introduced in either cell-bound form. Upon examination of the draining lymph nodes (LNs) during the first days of infection, virus-producing CD4+T cells predominated in control animals that received s.c. cell-free virus. In dramatic contrast, both SIV-positive macrophages and T cells were detected in the LNs of monkeys infected with cell-associated SIV. Therefore, although both cell-free and cell-associated viruses are infectious, the initial cells amplifying the virus differ. This may have important implications for the subsequent dissemination of infection and/or induction of antiretroviral immunity.
Protective Immunity to Rabbit Oral and Cutaneous Papillomaviruses by Immunization with Short Peptides of L2, the Minor Capsid Protein
Authors: Embers Monica E. , Budgeon Lynn R. , Pickel Martin , Christensen Neil D. 
Published: September 2002

ABSTRACT
The papillomavirus minor capsid protein, L2, has been shown to exhibit immunogenicity, whereby a variety of B-cell epitopes, predominantly in the amino terminus of L2, have been deduced. However, immunity to L2 in vivo has not been examined extensively. Notably, a common neutralization epitope for human papillomavirus (HPV) types 6 and 16 was mapped to amino acids (aa) 108 to 120. The objectives of this study were to derive antisera from rabbits using the corresponding sequences from rabbit viruses and to assess the ability of these peptides to protect against infection. Synthetic peptides consisting of two overlapping sequences each in the region of aa 94 to 122 of the rabbit oral (ROPV) and cottontail rabbit (CRPV) papillomaviruses were used to immunize rabbits. Rabbits were then infected with both ROPV and CRPV and monitored for the development of oral and cutaneous papillomas, respectively. Serum derived from rabbits immunized with either of the two peptides was shown to (i) react to purified L2 from the cognate virus, (ii) specifically recognize L2 within virus-infected cells, and (iii) neutralize virus in vitro. Following viral challenge, cutaneous papilloma growth was completely absent in rabbits immunized with either CRPV peptide. Likewise, ROPV peptide-immunized rabbits were protected from oral papillomatosis. Challenge of CRPV peptide-immune rabbits with the viral genome resulted in efficient papilloma growth, suggesting a neutralizing antibody-mediated mechanism of protection. These results afford in vivo evidence for the immunogenicity provided by a distinct region of L2 and further support previous evidence for the ability of this region to elicit antiviral immunity.
Identification of a G
2
Arrest Domain in the E1∧E4 Protein of Human Papillomavirus Type 16
Authors: Davy Clare E. , Jackson Deborah J. , Wang Qian , Raj Kenneth , Masterson Phillip J. , Fenner Nicola F. , Southern Shirley , Cuthill Scott , Millar Jonathan B. A. , Doorbar John 
Published: September 2002

ABSTRACT

Human papillomavirus type 16 (HPV16) is the most common cause of cervical carcinoma. Cervical cancer develops from low-grade lesions that support the productive stages of the virus life cycle. The 16E1∧E4 protein is abundantly expressed in such lesions and can be detected in cells supporting vegetative viral genome amplification. Using an inducible mammalian expression system, we have shown that 16E1∧E4 arrests HeLa cervical epithelial cells in G
2
. 16E1∧E4 also caused a G
2
arrest in SiHa, Saos-2 and
Saccharomyces pombe
cells and, as with HeLa cells, was found in the cytoplasm. However, whereas 16E1∧E4 is found on the keratin networks in HeLa and SiHa cells, in Saos-2 and
S. pombe
cells that lack keratins, 16E1∧E4 had a punctate distribution. Mutagenesis studies revealed a proline-rich region between amino acids 17 and 45 of 16E1∧E4 to be important for arrest. This region, which we have termed the “arrest domain,” contains a putative nuclear localization signal, a cyclin-binding motif, and a single cyclin-dependent kinase (Cdk) phosphorylation site. A single point mutation in the putative Cdk phosphorylation site (T23A) abolished 16E1∧E4-mediated G
2
arrest. Arrest did not involve proteins regulating the phosphorylation state of Cdc2 and does not appear to involve the activation of the DNA damage or incomplete replication checkpoint. G
2
arrest was also mediated by the E1∧E4 protein of HPV11, a low-risk mucosal HPV type that also causes cervical lesions. The E1∧E4 protein of HPV1, which is more distantly related to that of HPV16, did not cause G
2
arrest. We conclude that, like other papillomavirus proteins, 16E1∧E4 affects cell cycle progression and that it targets a conserved component of the cell cycle machinery.
Kinetics of Expression of Rhesus Monkey Rhadinovirus (RRV) and Identification and Characterization of a Polycistronic Transcript Encoding the RRV Orf50/Rta, RRV R8, and R8.1 Genes
Authors: DeWire Scott M. , McVoy Michael A. , Damania Blossom 
Published: September 2002

ABSTRACT
Rhesus monkey rhadinovirus (RRV) is a close relative of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8). RRV serves as an in vitro and an in vivo model for KSHV, and the mapping of its transcription program during lytic replication is significant since it represents de novo infection in the absence of stimulation with phorbol esters. Further, the RRV lytic system facilitates the making of recombinant viruses, and hence transcription profiling of the wild-type virus is important. Currently, the kinetics of lytic gene expression of RRV, the function of the RRV Orf50/Rta gene, and the presence of the RRV R8 and R8.1 genes are not known. This study details the transcription profile seen during RRV lytic replication and shows that RRV latency-associated nuclear antigen, viral FLIP (vFLIP), and vCyclin are transcribed during the RRV lytic phase. In addition, this study describes the identification of three new spliced products of the RRV Orf50, R8, and R8.1 genes, which are structural homologs of the KSHV Orf50, K8, and K8.1 genes, respectively. Characterization of the RRV Orf50 protein identifies it as a strong transcriptional transactivator capable of activating three early RRV promoters. Interestingly, the KSHV Orf50 transactivator can also activate these simian virus promoters, suggesting that there exists a conservation of gene function between the key transcription factors of KSHV and RRV.
A Single Mutation in the Carboxy Terminus of Reovirus Outer-Capsid Protein σ3 Confers Enhanced Kinetics of σ3 Proteolysis, Resistance to Inhibitors of Viral Disassembly, and Alterations in σ3 Structure
Authors: Wilson Gregory J. , Nason Emma L. , Hardy Charles S. , Ebert Daniel H. , Wetzel J. Denise , Venkataram Prasad B. V. , Dermody Terence S. 
Published: September 2002

ABSTRACT
Mammalian reoviruses undergo acid-dependent proteolytic disassembly within endosomes, resulting in formation of infectious subvirion particles (ISVPs). ISVPs are obligate intermediates in reovirus disassembly that mediate viral penetration into the cytoplasm. The initial biochemical event in the reovirus disassembly pathway is the proteolysis of viral outer-capsid protein σ3. Mutant reoviruses selected during persistent infection of murine L929 cells (PI viruses) demonstrate enhanced kinetics of viral disassembly and resistance to inhibitors of endocytic acidification and proteolysis. To identify sequences in σ3 that modulate acid-dependent and protease-dependent steps in reovirus disassembly, the σ3 proteins of wild-type strain type 3 Dearing; PI viruses L/C, PI 2A1, and PI 3-1; and four novel mutant σ3 proteins were expressed in insect cells and used to recoat ISVPs. Treatment of recoated ISVPs (rISVPs) with either of the endocytic proteases cathepsin L or cathepsin D demonstrated that an isolated tyrosine-to-histidine mutation at amino acid 354 (Y354H) enhanced σ3 proteolysis during viral disassembly. Yields of rISVPs containing Y354H in σ3 were substantially greater than those of rISVPs lacking this mutation after growth in cells treated with either acidification inhibitor ammonium chloride or cysteine protease inhibitor E64. Image reconstructions of electron micrographs of virus particles containing wild-type or mutant σ3 proteins revealed structural alterations in σ3 that correlate with the Y354H mutation. These results indicate that a single mutation in σ3 protein alters its susceptibility to proteolysis and provide a structural framework to understand mechanisms of σ3 cleavage during reovirus disassembly.
Identification and Characterization of Three Immunodominant Structural Proteins of Fowlpox Virus
Authors: Boulanger Denise , Green Philip , Jones Brenda , Henriquet Gwenn , Hunt Lawrence G. , Laidlaw Stephen M. , Monaghan Paul , Skinner Michael A. 
Published: September 2002

ABSTRACT
Genes encoding fowlpox virus (FWPV) structural proteins have been identified mainly by sequence homology with those from vaccinia virus (VACV), but little is known about the encoded proteins. Production of monoclonal antibodies (MAbs) against Poxine and HP1-440 (Munich) clone FP9 allowed the identification of three immunodominant FWPV proteins: the 39-kDa core protein (encoded by FPV168, homologous to VACV A4L), a 30- and 35-kDa protein doublet, and an abundant 63-kDa protein. The 30- and 35-kDa proteins are nonglycosylated, antigenically related proteins present in the intracellular mature virus membrane and localizing closely with the viral factories. N-terminal sequencing identified the 35-kDa protein as encoded by FPV140 (the FWPV homolog of VACV H3L). The 63-kDa protein forms covalently linked dimers and oligomers. It remained mainly insoluble upon detergent treatment of purified virus but did not localize closely with the viral factory. N-terminal sequencing was unsuccessful, suggesting N-terminal blocking. CNBr digestion generated a peptide encoded by FPV191, predicted to encode one of two FWPV A-type inclusion (ATI) proteins. The characteristics of the 63-kDa protein were inconsistent with published observations on cowpox or VACV ATI proteins (it appears to be essential). The 63-kDa protein, however, shares characteristics with both VACV p4c virus occlusion and 14-kDa fusion proteins. Gene assignment at the poxvirus ATI locus (between VACV A24R and A28L) is complicated by sequence redundancies and variations, often due to deletions and multiple frameshift mutations. The identity of FPV191 in relation to genes at this locus is discussed.
Flock House Virus RNA Polymerase Is a Transmembrane Protein with Amino-Terminal Sequences Sufficient for Mitochondrial Localization and Membrane Insertion
Authors: Miller David J. , Ahlquist Paul 
Published: September 2002

ABSTRACT

Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. Replication complexes of flock house virus (FHV), the best-studied alphanodavirus, are located on outer mitochondrial membranes in infected
Drosophila melanogaster
cells and are associated with the formation of membrane-bound spherules, similar to structures found for many other positive-strand RNA viruses. To further study FHV replication complex formation, we investigated the subcellular localization, membrane association, and membrane topology of protein A, the FHV RNA-dependent RNA polymerase, in the yeast
Saccharomyces cerevisiae
, a host able to support full FHV RNA replication and virion formation. Confocal immunofluorescence revealed that protein A localized to mitochondria in yeast, as in
Drosophila
cells, and that this mitochondrial localization was independent of viral RNA synthesis. Nycodenz gradient flotation and dissociation assays showed that protein A behaved as an integral membrane protein, a finding consistent with a predicted N-proximal transmembrane domain. Protease digestion and selective permeabilization after differential epitope tagging demonstrated that protein A was inserted into the outer mitochondrial membrane with the N terminus in the inner membrane space or matrix and that the C terminus was exposed to the cytoplasm. Flotation and immunofluorescence studies with deletion mutants indicated that the N-proximal region of protein A was important for both membrane association and mitochondrial localization. Gain-of-function studies with green fluorescent protein fusions demonstrated that the N-terminal 46 amino acids of protein A were sufficient for mitochondrial localization and membrane insertion. We conclude that protein A targets and anchors FHV RNA replication complexes to outer mitochondrial membranes, in part through an N-proximal mitochondrial localization signal and transmembrane domain.
Memory CD4
+
T Cells Are the Earliest Detectable Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Cells in the Female Genital Mucosal Tissue during HIV-1 Transmission in an Organ Culture System
Authors: Gupta Phalguni , Collins Kelly B. , Ratner Deena , Watkins Simon , Naus Gregory J. , Landers Daniel V. , Patterson Bruce K. 
Published: September 2002

ABSTRACT

The virologic and cellular factors that are involved in transmission of human immunodeficiency virus type 1 (HIV-1) across the female genital tissue are poorly understood. We have recently developed a human cervical tissue-derived organ culture model to study heterosexual transmission of HIV-1 that mimics the in vivo situation. Using this model we investigated the role of phenotypic characteristics of HIV-1 and identified the cell types that are first infected during transmission. Our data indicate that the cell-free R5 HIV-1 was more efficiently transmitted than cell-free X4 HIV-1. Cell-free and cell-associated HIV-1 had comparable transmission efficiency regardless of whether the virus was of R5 or X4 type. We have demonstrated that memory CD4
+
T cells and not Langerhans cells were the first HIV-1 RNA-positive cells detected at the epithelial-submucosal junction 6 h after virus exposure. Multicolor laser confocal microscopy demonstrated a globular distribution of HIV-1
gag-pol
mRNA in the cytoplasm, and the distribution of CD4 and the CD45RO isoform was irregular on the cellular membrane. At 96 h postinoculation, in addition to memory CD4
+
T cells, HIV-1 RNA-positive Langerhans cells and macrophages were also detected. The identification of CD4
+
T cells in the tissue at 6 h was confirmed by flow cytometric simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization assay on immune cells isolated from disaggregated tissue. Furthermore, PMPA {9-[2-(phosphonomethoxy)propyl] adenine}, an antiretroviral compound, and UC781, a microbicide, inhibited HIV-1 transmission across the mucosa, indicating the utility of the organ culture to screen topical microbicides for their ability to block sexual transmission of HIV-1.
Activation of Terminally Differentiated Human Monocytes/Macrophages by Dengue Virus: Productive Infection, Hierarchical Production of Innate Cytokines and Chemokines, and the Synergistic Effect of Lipopolysaccharide
Authors: Chen Yun-Chi , Wang Sheng-Yuan 
Published: September 2002

ABSTRACTDengue virus (DV) primarily infects blood monocytes (MO) and tissue macrophages (Mφ). We have shown in the present study that DV can productively infect primary human MO/Mφ regardless of the stage of cell differentiation. After DV infection, the in vitro-differentiated MO/Mφ secreted multiple innate cytokines and chemokines, including tumor necrosis factor alpha, alpha interferon (IFN-α), interleukin-1β (IL-1β), IL-8, IL-12, MIP-1α, and RANTES but not IL-6, IL-15, or nitric oxide. Secretion of these mediators was highlighted by distinct magnitude, onset, kinetics, duration, and induction potential. A chemokine-to-cytokine hierarchy was noted in the magnitude and induction potential of secretion, and a chemokine-to-cytokine-to-chemokine/Th1 cytokine cascade could be seen in the production kinetics. Furthermore, we found that terminally differentiated MO/Mφ cultured for more than 45 days could support productive DV infection and produce innate cytokines and chemokines, indicating that these mature cells were functionally competent in the context of a viral infection. In addition, DV replication in primary differentiated human MO/Mφ was enhanced and prolonged in the presence of lipopolysaccharide (LPS), and LPS-mediated synergistic production of IFN-α could be seen in DV-infected MO/Mφ. The secretion of innate cytokines and chemokines by differentiated MO/Mφ suggests that regional accumulation of these mediators may occur in various tissues to which DV has disseminated and may thus result in local inflammation. The LPS-mediated enhancement of virus replication and synergistic IFN-α production suggests that concurrent bacterial infection may modulate cytokine-mediated disease progression during DV infection.
Mutagenic Stabilization and/or Disruption of a CD4-Bound State Reveals Distinct Conformations of the Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein
Authors: Xiang Shi-Hua , Kwong Peter D. , Gupta Rishi , Rizzuto Carlo D. , Casper David J. , Wyatt Richard , Wang Liping , Hendrickson Wayne A. , Doyle Michael L. , Sodroski Joseph 
Published: September 2002

ABSTRACTThe human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein is conformationally flexible. Upon binding to the host cell receptor CD4, gp120 assumes a conformation that is recognized by the second receptor, CCR5 and/or CXCR4, and by the CD4-induced (CD4i) antibodies. Guided by the X-ray crystal structure of a gp120-CD4-CD4i antibody complex, we introduced changes into gp120 that were designed to stabilize or disrupt this conformation. One mutant, 375 S/W, in which the tryptophan indole group is predicted to occupy the Phe 43 cavity in the gp120 interior, apparently favors a gp120 conformation closer to that of the CD4-bound state. The 375 S/W mutant was recognized as well as or better than wild-type gp120 by CD4 and CD4i antibodies, and the large decrease in entropy observed when wild-type gp120 bound CD4 was reduced for the 375 S/W mutant. The recognition of the 375 S/W mutant by CD4BS antibodies, which are directed against the CD4-binding region of gp120, was markedly reduced compared with that of the wild-type gp120. Compared with the wild-type virus, viruses with the 375 S/W envelope glycoproteins were resistant to neutralization by IgG1b12, a CD4BS antibody, were slightly more sensitive to soluble CD4 neutralization and were neutralized more efficiently by the 2G12 antibody. Another mutant, 423 I/P, in which the gp120 bridging sheet was disrupted, did not bind CD4, CCR5, or CD4i antibodies, even though recognition by CD4BS antibodies was efficient. These results indicate that CD4BS antibodies recognize conformations of gp120 different from that recognized by CD4 and CD4i antibodies.