bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Anatomy of Herpes Simplex Virus 1 Overlapping Genes Encoding Infected-Cell Protein 22 and U
S
1.5 Protein
Authors: Ogle William O. , Roizman Bernard 
Published: December 2019

ABSTRACT

Earlier studies have shown that (i) the coding domain of the α22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and a protein, U
S
1.5, which is initiated from methionine 147 of ICP22 and which is colinear with the remaining portion of that protein; (ii) posttranslational processing of ICP22 mediated largely by the viral protein kinase U
L
13 yields several isoforms differing in electrophoretic mobility; and (iii) mutants lacking the carboxyl-terminal half of the ICP22 and therefore ΔU
S
1.5 are avirulent and fail to express normal levels of subsets of both α (e.g., ICP0) or γ
2
(e.g., U
S
11 and U
L
38) proteins. We have generated and analyzed two sets of recombinant viruses. The first lacked portions of or all of the sequences expressed solely by ICP22. The second set lacked 10 to 40 3′-terminal codons of ICP22 and U
S
1.5. The results were as follows. (i) In cells infected with mutants lacking amino-terminal sequences, translation initiation begins at methionine 147. The resulting protein cannot be differentiated in mobility from authentic U
S
1.5, and its posttranslational processing is mediated by the U
L
13 protein kinase. (ii) Expression of U
S
11 and U
L
38 genes by mutants carrying only the U
S
1.5 gene is similar to that of wild-type parent virus. (iii) Mutants which express only U
S
1.5 protein are avirulent in mice. (iv) The coding sequences Met147 to Met171 are essential for posttranslational processing of the U
S
1.5 protein. (v) ICP22 made by mutants lacking 15 or fewer of the 3′-terminal codons are posttranslationally processed whereas those lacking 18 or more codons are not processed. (vi) Wild-type and mutant ICP22 proteins localized in both nucleus and cytoplasm irrespective of posttranslational processing. We conclude that ICP22 encodes two sets of functions, one in the amino terminus unique to ICP22 and one shared by ICP22 and U
S
1.5. These functions are required for viral replication in experimental animals. U
S
1.5 protein must be posttranslationally modified by the U
L
13 protein kinase to enable expression of a subset of late genes exemplified by U
L
38 and U
S
11. Posttranslational processing is determined by two sets of sequences, at the amino terminus and at the carboxyl terminus of U
S
1.5, respectively, a finding consistent with the hypothesis that both domains interact with protein partners for specific functions.
Long-Term Episomal Maintenance of Bovine Papillomavirus Type 1 Plasmids Is Determined by Attachment to Host Chromosomes, Which Is Mediated by the Viral E2 Protein and Its Binding Sites
Authors: Ilves Ivar , Kivi Sirje , Ustav Mart 
Published: December 2019

ABSTRACT

Papillomavirus genomes are stably maintained as extrachromosomal nuclear plasmids in dividing host cells. To address the mechanisms responsible for stable maintenance of virus, we examined nuclear compartmentalization of plasmids containing the full-length upstream regulatory region (URR) from the bovine papillomavirus type 1 (BPV1) genome. We found that these plasmids are tightly associated with the nuclear chromatin both in the stable cell lines that maintain episomal copies of the plasmids and in transiently transfected cells expressing the viral E1 and E2 proteins. Further analysis of viral factors revealed that the E2 protein in
trans
and its multiple binding sites in
cis
are both necessary and sufficient for the chromatin attachment of the plasmids. On the other hand, the BPV1 URR-dependent plasmid replication and chromatin attachment processes are clearly independent of each other. The ability of the plasmids to stably maintain episomes correlates clearly with their chromatin association function. These data suggest that viral E2 protein-mediated attachment of BPV1 genomes to the host cell chromatin could provide a mechanism for the coupling of viral genome multiplication and partitioning to the host cell cycle during viral latent infection.
Cellular Elongation Factor 1δ Is Modified in Cells Infected with Representative Alpha-, Beta-, or Gammaherpesviruses
Authors: Kawaguchi Yasushi , Matsumura Tomio , Roizman Bernard , Hirai Kanji 
Published: December 2019

ABSTRACT

Earlier reports (Y. Kawaguchi, R. Bruni, and B. Roizman, J. Virol. 71:1019–1024, 1997; Y. Kawaguchi, C. Van Sant, and B. Roizman, J. Virol. 72:1731–1736, 1998) showed that herpes simplex virus 1 (HSV-1) infection causes the hyperphosphorylation of translation elongation factor 1δ (EF-1δ) and that the modification of EF-1δ is the consequence of direct phosphorylation by a viral protein kinase encoded by the U
L
13 gene of HSV-1. The U
L
13 gene is conserved in members of all herpesvirus subfamilies. Here we report the following. (i) In various mammalian cells, accumulation of the hyperphosphorylated form of EF-1δ is observed after infection with alpha-, beta-, and gammaherpesviruses, including HSV-2, feline herpesvirus 1, pseudorabiesvirus, bovine herpesvirus 1, human cytomegalovirus (HCMV), and equine herpesvirus 2. (ii) In human lung fibroblast cells infected with recombinant HSV-1 lacking the U
L
13 gene, the hypophosphorylated form of EF-1δ is a minor species, whereas the amount of the hyperphosphorylated form of EF-1δ significantly increases in cells infected with the recombinant HSV-1 in which U
L
13 had been replaced by HCMV U
L
97, a homologue of U
L
13. These results indicate that the posttranslational modification of EF-1δ is conserved herpesvirus function and the U
L
13 homologues may be responsible for the universal modification of the translation factor.
Redox and Activation Status of Monocytes from Human Immunodeficiency Virus-Infected Patients: Relationship with Viral Load
Authors: Elbim C. , Pillet S. , Prevost M. H. , Preira A. , Girard P. M. , Rogine N. , Matusani H. , Hakim J. , Israel N. , Gougerot-Pocidalo M. A. 
Published: December 2019

ABSTRACTMonocytes are precursors of tissue macrophages, which are major targets of human immunodeficiency virus type 1 (HIV-1) infection. Although few blood monocytes are infected, their resulting activation could play a key role in the pathogenesis of HIV disease by modulating their transendothelial migration and inducing the production of reactive oxygen species (ROS). ROS participate in chronic inflammation, HIV replication, and the apoptosis of immune system cells seen in HIV-infected subjects. Published data on monocyte activation are controversial, possibly because most studies have involved monocytes isolated from their blood environment by various procedures that may alter cell responses. We therefore used flow cytometry to study, in whole blood, the activation and redox status of monocytes from HIV-infected patients at different stages of the disease. We studied the expression of adhesion molecules, actin polymerization, and cellular levels of H2O2, Bcl-2, and thioredoxin. Basal H2O2production correlated with viral load and was further enhanced by bacterialN-formyl peptides and endotoxin. The enhanced H2O2production by monocytes from asymptomatic untreated patients with CD4+cell counts above 500/μl was associated with a decrease in the levels of Bcl-2 and thioredoxin. In contrast, in patients with AIDS, Bcl-2 levels returned to normal and thioredoxin levels were higher than in healthy controls. Restoration of these antioxidant and antiapoptotic molecules might explain, at least in part, why monocyte numbers remain relatively stable throughout the disease. Alterations of adhesion molecule expression and increased actin polymerization could play a role in transendothelial migration of these activated monocytes.
Human CD46 Enhances Nitric Oxide Production in Mouse Macrophages in Response to Measles Virus Infection in the Presence of Gamma Interferon: Dependence on the CD46 Cytoplasmic Domains
Authors: Hirano Akiko , Yang Ziping , Katayama Yuko , Korte-Sarfaty Jennifer , Wong Timothy C. 
Published: December 2019

ABSTRACTCD46 is a transmembrane complement regulatory protein widely expressed on nucleated human cells. Laboratory-adapted strains of measles virus (MV) bind to the extracellular domains of CD46 to enter human cells. The cytoplasmic portion of CD46 consists of a common juxtamembrane region and different distal sequences called Cyt1 and Cyt2. The biological functions of these cytoplasmic sequences are unknown. In this study, we show that expression of human CD46 with the Cyt1 cytoplasmic domain in mouse macrophages enhances production of nitric oxide (NO) in response to MV infection in the presence of gamma interferon (IFN-γ). Human CD46 does not increase the basal levels of NO production in mouse macrophages and does not augment NO production induced by double-stranded polyribonucleotides. Replacing the cytoplasmic domain of human CD46 with Cyt2 reduces MV and IFN-γ-induced NO production in mouse macrophages. Deleting the entire cytoplasmic domains of human CD46 does not prevent MV infection but markedly attenuates NO production in response to MV and IFN-γ. Mouse macrophages expressing a tailless human CD46 mutant are more susceptible to MV infection and produce 2 to 3 orders of magnitude more infectious virus than mouse macrophages expressing human CD46 with intact cytoplasmic domains. These results reveal a novel function of CD46 dependent on the cytoplasmic domains (especially Cyt1), which augments NO production in macrophages. These findings may have significant implications for roles of CD46 in innate immunity and MV pathogenesis.
Molecular Requirements for Human Immunodeficiency Virus Type 1 Plus-Strand Transfer: Analysis in Reconstituted and Endogenous Reverse Transcription Systems
Authors: Wu Tiyun , Guo Jianhui , Bess Julian , Henderson Louis E. , Levin Judith G. 
Published: December 2019

ABSTRACT

We have developed a reconstituted system which models the events associated with human immunodeficiency virus type 1 (HIV-1) plus-strand transfer. These events include synthesis of plus-strand strong-stop DNA [(+) SSDNA] from a minus-strand DNA donor template covalently attached to human tRNA
3
Lys
, tRNA primer removal, and annealing of (+) SSDNA to the minus-strand DNA acceptor template. Termination of (+) SSDNA synthesis at the methyl A (nucleotide 58) near the 3′ end of tRNA
3
Lys
reconstitutes the 18-nucleotide primer binding site (PBS). Analysis of (+) SSDNA synthesis in vitro and in HIV-1 endogenous reactions indicated another major termination site: the pseudouridine at nucleotide 55. In certain HIV-1 strains, complementarity between nucleotides 56 to 58 and the first three bases downstream of the PBS could allow all of the (+) SSDNA products to be productively transferred. Undermodification of the tRNA may be responsible for termination beyond the methyl A. In studies of tRNA removal, we find that initial cleavage of the 3′ rA by RNase H is not sufficient to achieve successful strand transfer. The RNA-DNA hybrid formed by the penultimate 17 bases of tRNA still annealed to (+) SSDNA must also be destabilized. This can occur by removal of additional 3′-terminal bases by RNase H (added either in
cis
or
trans
). Alternatively, the nucleic acid chaperone activity of nucleocapsid protein (NC) can catalyze this destabilization. NC stimulates annealing of the complementary PBS sequences in (+) SSDNA and the acceptor DNA template. Reverse transcriptase also promotes annealing but to a lesser extent than NC.
Quantitative Analysis of Latent Human Cytomegalovirus
Authors: Slobedman Barry , Mocarski Edward S. 
Published: December 2019

ABSTRACT
Cytomegalovirus latency depends on an interaction with hematopoietic cells in bone marrow and peripheral blood. The distribution of viral DNA was investigated by PCR-driven in situ hybridization (PCR-ISH), and the number of viral genomes per cell was estimated by quantitative competitive PCR during both experimental and natural latent infection. During experimental latent infection of cultured granulocyte-macrophage progenitors, the viral genome was detected in >90% of cells at a copy number of 1 to 8 viral genomes per cell. During natural infection, viral genomes were detected in 0.004 to 0.01% of mononuclear cells from granulocyte colony-stimulating factor-mobilized peripheral blood or bone marrow from seropositive donors, at a copy number of 2 to 13 genomes per infected cell. When evaluated by reverse transcription–PCR-ISH, only a small proportion of experimentally infected cells (approximately 2%) had detectable latent transcripts. This investigation identifies the small percentage of bone marrow-derived mononuclear cells that become latently infected during natural infection and suggests that latency may proceed in some cells that fail to encode currently identified latent transcripts.
Characterization of a Major Neutralizing Epitope on Human Papillomavirus Type 16 L1
Authors: White Wendy I. , Wilson Susan D. , Palmer-Hill Frances J. , Woods Robert M. , Ghim Shin-je , Hewitt Lisa A. , Goldman Daniel M. , Burke Steven J. , Jenson A. Bennett , Koenig Scott , Suzich JoAnn A. 
Published: December 2019

ABSTRACT
Persistent infection with human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Neutralizing epitopes present on the major coat protein, L1, have not been well characterized, although three neutralizing monoclonal antibodies (MAbs) had been identified by using HPV-16 pseudovirions (R. B. Roden et al., J. Virol. 71:6247–6252, 1997). Here, two of these MAbs (H16.V5 and H16.E70) were demonstrated to neutralize authentic HPV-16 in vitro, while the third (H16.U4) did not. Binding studies were conducted with the three MAbs and virus-like particles (VLPs) composed of the reference L1 sequence (114K) and three variant L1 sequences: Rochester-1k (derived from viral stock DNA), GU-1 (derived from cervical biopsy DNA), and GU-2 (derived from biopsy DNA, but containing some sequence changes likely to be artifactual). While all three MAbs bound to 114K and Rochester-1k VLPs, GU-1 VLPs were not recognized by H16.E70, and both H16.E70 and H16.V5 failed to bind to GU-2 VLPs. Site-directed mutagenesis was used to replace disparate amino acids in the GU-2 L1 with those found in the 114K L1. Alteration of the amino acid at position 50, from L to F, completely restored H16.V5 binding and partially restored H16.E70 binding, while complete restoration of H16.E70 binding occurred with GU-2 VLPs containing both L50F and T266A alterations. Immunization of mice with L1 variant VLPs revealed that GU-2 VLPs were poorly immunogenic. The L50F mutant of GU-2 L1, in which the H16.V5 epitope was restored, elicited HPV-16 antibody responses comparable to those obtained with 114K VLPs. These results demonstrate the importance of the H16.V5 epitope in the generation of potent HPV-16 neutralizing antibody responses.
Interactions of the Papovavirus DNA Replication Initiator Proteins, Bovine Papillomavirus Type 1 E1 and Simian Virus 40 Large T Antigen, with Human Replication Protein A
Authors: Han YuFeng , Loo Yueh-Ming , Militello Kevin T. , Melendy Thomas 
Published: December 2019

ABSTRACT

Papovaviruses utilize predominantly cellular DNA replication proteins to replicate their own viral genomes. To appropriate the cellular DNA replication machinery, simian virus 40 (SV40) large T antigen (Tag) binds to three different cellular replication proteins, the DNA polymerase α-primase complex, the replication protein A (RPA) complex, and topoisomerase I. The functionally similar papillomavirus E1 protein has also been shown to bind to the DNA polymerase α-primase complex. Enzyme-linked immunoassay-based protein interaction assays and protein affinity pull-down assays were used to show that the papillomavirus E1 protein also binds to the cellular RPA complex in vitro. Furthermore, SV40 Tag was able to compete with bovine papillomavirus type 1 E1 for binding to RPA. Each of the three RPA subunits was individually overexpressed in
Escherichia coli
as a soluble fusion protein. These fusion proteins were used to show that the E1-RPA and Tag-RPA interactions are primarily mediated through the 70-kDa subunit of RPA. These results suggest that different viruses have evolved similar mechanisms for taking control of the cellular DNA replication machinery.
Papillomavirus Capsid Protein Expression Level Depends on the Match between Codon Usage and tRNA Availability
Authors: Zhou Jian , Liu Wen Jun , Peng Shi Wen , Sun Xiao Yi , Frazer Ian 
Published: December 2019

ABSTRACT

Translation of mRNA encoding the L1 and L2 capsid proteins of papillomavirus (PV) is restricted in vivo to differentiated epithelial cells, although transcription of the L1 and L2 late genes occurs more widely. The codon composition of PV late genes is quite different from that of most mammalian genes. To test the possibility that PV late gene codon composition determines the efficiency of PV late gene expression in some cell types, synthetic bovine papillomavirus type 1 (BPV1) late genes were constructed with codon composition modified to resemble the typical mammalian gene. Expression of these genes from a strong promoter in Cos-1 cells was compared with expression of wild-type BPV1 late genes from the same promoter. Both unmodified and modified PV late genes were transcribed in Cos-1 cells, but only the codon-modified genes were translated. In vitro translation of wild-type but not synthetic BPV1 L1 mRNA was markedly enhanced by addition of aminoacyl-tRNAs. Codon composition thus limits BPV1 late gene translation in Cos-1 cells, and this limitation can be overcome by modification of the codon composition of the genes or by provision of excess tRNA. Replacement of codons in the green fluorescent protein (
gfp
) gene with those frequently used in PV late genes did not alter
gfp
transcription in Cos-1 cells but almost abolished translation, supporting the hypothesis that the observed differences in efficiency of translation of modified and unmodified PV capsid genes were related to codon usage rather than mRNA structure. As tRNA populations vary within and between tissues in the same eukaryotic organism, we speculate that matching of tRNA availability to codon usage may be one determinant of the restriction of expression of PV late genes to differentiated epithelium.
Rescue of Newcastle Disease Virus from Cloned cDNA: Evidence that Cleavability of the Fusion Protein Is a Major Determinant for Virulence
Authors: Peeters Ben P. H. , de Leeuw Olav S. , Koch Guus , Gielkens Arno L. J. 
Published: December 2019

ABSTRACT
A full-length cDNA clone of Newcastle disease virus (NDV) vaccine strain LaSota was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Transfection of this plasmid into cells that were infected with a recombinant fowlpoxvirus that expressed T7 RNA polymerase, resulted in the synthesis of antigenomic NDV RNA. This RNA was replicated and transcribed by the viral NP, P, and L proteins, which were expressed from cotransfected plasmids. After inoculation of the transfection supernatant into embryonated specific-pathogen-free eggs, infectious virus derived from the cloned cDNA was recovered. By introducing three nucleotide changes in the cDNA, we generated a genetically tagged derivative of the LaSota strain in which the amino acid sequence of the protease cleavage site (GGRQGR↓L) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR↓F). Pathogenicity tests in day-old chickens showed that the strain derived from the unmodified cDNA was completely nonvirulent (intracerebral pathogenicity index [ICPI] = 0.00). However, the strain derived from the cDNA in which the protease cleavage site was modified showed a dramatic increase in virulence (ICPI = 1.28 out of a possible maximum of 2.0). Pulse-chase labeling of cells infected with the different strains followed by radioimmunoprecipitation of the F protein showed that the efficiency of cleavage of the F0 protein was greatly enhanced by the amino acid replacements. These results demonstrate that genetically modified NDV can be recovered from cloned cDNA and confirm the supposition that cleavage of the F0 protein is a key determinant in virulence of NDV.
Dynamics of Nontypical Apoptotic Morphological Changes Visualized by Green Fluorescent Protein in Living Cells with Infectious Pancreatic Necrosis Virus Infection
Authors: Hong Jiann-Ruey , Lin Tai-Lang , Yang Jer-Yen , Hsu Ya-Li , Wu Jen-Leih 
Published: December 2019

ABSTRACT
Morphologically, apoptotic cells are characterized by highly condensed membrane blebbing and formation of apoptotic bodies. Recently, we reported that apoptosis precedes necrosis in a fish cell line infected with infectious pancreatic necrosis virus (IPNV). In the present study, we tested the possibility that nontypical apoptosis is a component of IPNV-induced fish cell death. A variant type of green fluorescent protein (EGFP) was expressed in a fish cell line such that EGFP served as a protein marker for visualizing dynamic apoptotic cell morphological changes and for tracing membrane integrity changes during IPNV infection. Direct morphological changes were visualized by fluorescence microscopy by EGFP in living cells infected with IPNV. The nontypical apoptotic morphological change stage occurred during the pre-late stage (6 to 7 h postinfection). Nontypical apoptotic features, including highly condensed membrane blebbing, occurred during the middle apoptotic stage. At the pre-late apoptotic stage, membrane vesicles quickly formed, blebbed, and were finally pinched off from the cell membrane. At the same time, at this pre-late apoptotic stage, apoptotic cells formed unique small holes in their membranes that ranged from 0.39 to 0.78 μm according to examination by scanning electron microscopy and immunoelectron microscopy. Quantitation of the intra- and extracellular release of EGFP by CHSE-214-EGFP cells after IPNV infection was done by Western blotting and fluorometry. Membrane integrity was quickly lost during the late apoptotic stage (after 8 h postinfection), and morphological change and membrane integrity loss could be prevented and blocked by treatment with apoptosis inhibitors such as cycloheximide, genistein, and EDTA before IPNV infection. Together, these findings show the apoptotic features at the onset of pathology in host cells (early and middle apoptotic stages), followed secondarily by nontypical apoptosis (pre-late apoptotic stage) and then by postapoptotic necrosis (late apoptotic stage), of a fish cell line. Our results demonstrate that nontypical apoptosis is a component of IPNV-induced fish cell death.