bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Role of Hepatitis C Virus Chimeric Glycoproteins in the Infectivity of Pseudotyped Virus
Authors: Lagging L. Martin , Meyer Keith , Owens Randall J. , Ray Ranjit 
Published: December 2019

ABSTRACTThe putative envelope glycoproteins of hepatitis C virus (HCV) likely play an important role in the initiation of viral infection. Available information suggests that the genomic regions encoding the putative envelope glycoproteins, when expressed as recombinant proteins in mammalian cells, largely accumulate in the endoplasmic reticulum. In this study, genomic regions which include the putative ectodomain of the E1 (amino acids 174 to 359) and E2 (amino acids 371 to 742) glycoproteins were appended to the transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein. This provided a membrane anchor signal and the VSV incorporation signal at the carboxy termini of the E1 and E2 glycoproteins. The chimeric gene constructs exhibited expression of the recombinant proteins on the cell surface in a transient expression assay. When infected with a temperature-sensitive VSV mutant (ts045) and grown at the nonpermissive temperature (40.5°C), cells transiently expressing the E1 or E2 chimeric glycoprotein generated VSV/HCV pseudotyped virus. The resulting pseudotyped virus generated from E1 or E2 surprisingly exhibited the ability to infect mammalian cells and sera derived from chimpanzees immunized with the homologous HCV envelope glycoproteins neutralized pseudotyped virus infectivity. Results from this study suggested a potential functional role for both the E1 and E2 glycoproteins in the infectivity of VSV/HCV pseudotyped virus in mammalian cells. These observations further suggest the importance of using both viral glycoproteins in a candidate subunit vaccine and the potential for using a VSV/HCV pseudotyped virus to determine HCV neutralizing antibodies.
Mutations in Rotavirus Nonstructural Glycoprotein NSP4 Are Associated with Altered Virus Virulence
Authors: Zhang Mingdong , Zeng Carl Q.-Y. , Dong Yanjie , Ball Judith M. , Saif Linda J. , Morris Andrew P. , Estes Mary K. 
Published: December 2019

ABSTRACT

Rotaviruses are major pathogens causing life-threatening dehydrating gastroenteritis in children and animals. One of the nonstructural proteins, NSP4 (encoded by gene 10), is a transmembrane, endoplasmic reticulum-specific glycoprotein. Recently, our laboratory has shown that NSP4 causes diarrhea in 6- to 10-day-old mice by functioning as an enterotoxin. To confirm the role of NSP4 in rotavirus pathogenesis, we sequenced gene 10 from two pairs of virulent and attenuated porcine rotaviruses, the OSU and Gottfried strains. Comparisons of the NSP4 sequences from these two pairs of rotaviruses suggested that structural changes between amino acids (aa) 131 and 140 are important in pathogenesis. We next expressed the cloned gene 10 from the OSU virulent (OSU-v) and OSU attenuated (OSU-a) viruses by using the baculovirus expression system and compared the biological activities of the purified proteins. NSP4 from OSU-v virus increased intracellular calcium levels over 10-fold in intestinal cells when added exogenously and 6-fold in insect cells when expressed endogenously, whereas NSP4 from OSU-a virus had little effect. NSP4 from OSU-v caused diarrhea in 13 of 23 neonatal mice, while NSP4 from OSU-a caused disease in only 4 of 25 mice (
P
< 0.01). These results suggest that avirulence is associated with mutations in NSP4. Results from site-directed mutational analyses showed that mutated OSU-v NSP4 with deletion or substitutions in the region of aa 131 to 140 lost its ability to increase intracellular calcium levels and to induce diarrhea in neonatal mice, confirming the importance of amino acid changes from OSU-v NSP4 to OSU-a NSP4 in the alteration of virus virulence.
Hepatitis C Virus Core Protein Binds to the Cytoplasmic Domain of Tumor Necrosis Factor (TNF) Receptor 1 and Enhances TNF-Induced Apoptosis
Authors: Zhu Nongliao , Khoshnan Ali , Schneider Robert , Matsumoto Masayuki , Dennert Gunther , Ware Carl , Lai Michael M. C. 
Published: December 2019

ABSTRACT

The hepatitis C virus (HCV) core protein is known to be a multifunctional protein, besides being a component of viral nucleocapsids. Previously, we have shown that the core protein binds to the cytoplasmic domain of lymphotoxin β receptor, which is a member of tumor necrosis factor receptor (TNFR) family. In this study, we demonstrated that the core protein also binds to the cytoplasmic domain of TNFR 1. The interaction was demonstrated both by glutathione
S
-transferase fusion protein pull-down assay in vitro and membrane flotation method in vivo. Both the in vivo and in vitro binding required amino acid residues 345 to 407 of TNFR 1, which corresponds to the “death domain” of this receptor. We have further shown that stable expression of the core protein in a mouse cell line (BC10ME) or human cell lines (HepG2 and HeLa cells) sensitized them to TNF-induced apoptosis, as determined by the TNF cytotoxicity or annexin V apoptosis assay. The presence of the core protein did not alter the level of TNFR 1 mRNA in the cells or expression of TNFR 1 on the cell surface, suggesting that the sensitization of cells to TNF by the viral core protein was not due to up-regulation of TNFR 1. Furthermore, we observed that the core protein blocked the TNF-induced activation of RelA/NF-κB in murine BC10ME cells, thus at least partially accounting for the increased sensitivity of BC10ME cells to TNF. However, NF-κB activation was not blocked in core protein-expressing HeLa or HepG2 cells, implying another mechanism of TNF sensitization by core protein. These results together suggest that the core protein can promote cell death during HCV infection via TNF signaling pathways possibly as a result of its interaction with the cytoplasmic tail of TNFR 1. Therefore, TNF may play a role in HCV pathogenesis.
Scaffold Attachment Region-Mediated Enhancement of Retroviral Vector Expression in Primary T Cells
Authors: Agarwal Manju , Austin Timothy W. , Morel Franck , Chen Jingyi , Böhnlein Ernst , Plavec Ivan 
Published: December 2019

ABSTRACT

We have studied retroviral transgene expression in primary human lymphocytes. Our data demonstrate that transgene expression is high in activated primary CD4
+
T cells but significantly decreased in mitotically quiescent cells. Incorporation of a DNA fragment from the scaffold attachment region (SAR) of the human beta interferon gene into the vector improved transgene expression, particularly in quiescent cells. The SAR element functioned in an orientation-dependent manner and enhanced expression of Moloney murine leukemia virus- and murine embryonic stem cell-based vectors. Clonal analysis of transduced T cells showed that the SAR sequence did not confer position-independent expression on a transgene but rather prevented the decrease of expression when cells became quiescent. The SAR sequence also enhanced transgene expression in T cells generated from retrovirally transduced CD34-enriched hematopoietic progenitor-stem cells in a SCID-hu thymus-liver mouse model. We have used the SAR-containing retroviral vector to express the RevM10 gene, a
trans
-dominant mutant of the human immunodeficiency virus type 1 (HIV-1) Rev gene. Compared to a standard retroviral vector, the SAR-containing vector was up to 2 orders of magnitude more efficient in inhibiting replication of the HIV-1 virus in infected CD4
+
peripheral blood lymphocyte populations in vitro. This is the first demonstration that SAR elements can be used to improve retroviral vector expression in human primary T cells.
Cell Cycle Dysregulation by Human Cytomegalovirus: Influence of the Cell Cycle Phase at the Time of Infection and Effects on Cyclin Transcription
Authors: Salvant Bryan S. , Fortunato Elizabeth A. , Spector Deborah H. 
Published: December 2019

ABSTRACT

Human cytomegalovirus (HCMV) infection inhibits cell cycle progression and alters the expression of cyclins E, A, and B (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697–6704, 1995). In this study, we examined cell cycle progression, cyclin gene expression, and early viral events when the infection was initiated at different points in the cell cycle (G
0
, G
1
, and S). In all cases, infection led to cell cycle arrest. Cells infected in G
0
or G
1
phase also showed a complete or partial absence, respectively, of cellular DNA synthesis at a time when DNA synthesis occurred in the corresponding mock-infected cells. In contrast, when cells were infected near or during S phase, many cells were able to pass through S phase and undergo mitosis prior to cell cycle arrest. S-phase infection also produced a delay in the appearance of the viral cytopathic effect and in the synthesis of immediate-early and early proteins. Labeling of cells with bromodeoxyuridine immediately prior to HCMV infection in S phase revealed that viral protein expression occurred primarily in cells which were not engaged in DNA synthesis at the time of infection. The viral-mediated induction of cyclin E, maintenance of cyclin-B protein levels, and inhibitory effects on the accumulation of cyclin A were not significantly affected when infection occurred during different phases of the cell cycle (G
0
, G
1
, and S). However, there was a delay in the observed inhibition of cyclin A in cells infected during S phase. This finding was in accord with the pattern of cell cycle progression and delay in viral gene expression associated with S-phase infection. Analysis of the mRNA revealed that the effects of the virus on cyclin E and cyclin A, but not on cyclin B, were primarily at the transcriptional level.
The Cys-Rich Region of Hepatitis A Virus Cellular Receptor 1 Is Required for Binding of Hepatitis A Virus and Protective Monoclonal Antibody 190/4
Authors: Thompson Peter , Lu Jinhua , Kaplan Gerardo G. 
Published: December 2019

ABSTRACT

The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in
Escherichia coli
fused to glutathione
S
-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-
N
-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1− cells) was undetectable. HAV-infected cr5 and mut2 cells but not mut1, mut3, d1−, and DR2 cells developed the characteristic cytoplasmic granular fluorescence of HAV-infected cells. These results indicate that the Cys-rich region of havcr-1 and its first N-glycosylation site are required for binding of protective MAb 190/4 and HAV receptor function.
Involvement of Endoplasmic Reticulum Chaperones in the Folding of Hepatitis C Virus Glycoproteins
Authors: Choukhi Amélie , Ung Sophana , Wychowski Czeslaw , Dubuisson Jean 
Published: December 2019

ABSTRACT
The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2) which interact noncovalently to form a heterodimer (E1-E2). During the folding and assembly of HCV glycoproteins, a large portion of these proteins are trapped in aggregates, reducing the efficiency of native E1-E2 complex assembly. To better understand this phenomenon and to try to increase the efficiency of HCV glycoprotein folding, endoplasmic reticulum chaperones potentially interacting with these proteins were studied. Calnexin, calreticulin, and BiP were shown to interact with E1 and E2, whereas no interaction was detected between GRP94 and HCV glycoproteins. The association of HCV glycoproteins with calnexin and calreticulin was faster than with BiP, and the kinetics of interaction with calnexin and calreticulin were very similar. However, calreticulin and BiP interacted preferentially with aggregates whereas calnexin preferentially associated with monomeric forms of HCV glycoproteins or noncovalent complexes. Tunicamycin treatment inhibited the binding of HCV glycoproteins to calnexin and calreticulin, indicating the importance of N-linked oligosaccharides for these interactions. The effect of the co-overexpression of each chaperone on the folding of HCV glycoproteins was also analyzed. However, the levels of native E1-E2 complexes were not increased. Together, our data suggest that calnexin plays a role in the productive folding of HCV glycoproteins whereas calreticulin and BiP are probably involved in a nonproductive pathway of folding.
Simian Immunodeficiency Virus Replicates to High Levels in Sooty Mangabeys without Inducing Disease
Authors: Rey-Cuillé Marie-Anne , Berthier Jean-Luc , Bomsel-Demontoy Marie-Claude , Chaduc Yves , Montagnier Luc , Hovanessian Ara G. , Chakrabarti Lisa A. 
Published: December 2019

ABSTRACT

A serologic survey of primates living in a French zoo allowed identification of three cases of infection with simian immunodeficiency virus in sooty mangabeys (
Cercocebus atys
) (SIVsm). Viral isolates, which were designated SIVsmFr66, SIVsmFr74, and SIVsmFr85, were obtained after short-term culture of mangabey lymphoid cells. Phylogenetic analysis of
gag
and
env
sequences amplified directly from mangabey tissues showed that the three SIVsmFr were genetically close and that they constituted a new subtype within the diverse SIVsm–SIVmac–human immunodeficiency virus type 2 (HIV-2) group. We could reconstruct the transmission events that likely occurred in 1986 between the three animals and evaluate the divergence of SIVsmFr sequences since transmission. The estimated rate of mutation fixation was 6 × 10
−3
substitutions per site per year, which was as high as the rate found for SIVmac infection in macaques. These data indicated that SIVsmFr replicated at a high rate in mangabeys, despite the nonpathogenic character of infection in this host. The viral load evaluated by competitive PCR reached 20,000 viral DNA copies per 10
6
lymph node cells. In addition, productively infected cells were readily detected in mangabey lymphoid tissues by in situ hybridization. The amounts of viral RNA in plasma ranged from 10
5
to 10
7
copies per ml. The cell-associated and plasma viral loads were as high as those seen in susceptible hosts (humans or macaques) during the asymptomatic stage of HIV or SIVmac infections. Thus, the lack of pathogenicity of SIVsm for its natural host cannot be explained by limited viral replication or by tight containment of viral production.
Carbohydrates Facilitate Correct Disulfide Bond Formation and Folding of Rotavirus VP7
Authors: Mirazimi Ali , Svensson Lennart 
Published: December 2019

ABSTRACT
It is well established that glycosylation is essential for assembly of enveloped viruses, but no information is yet available as to the function of carbohydrates on the nonenveloped but glycosylated rotavirus. We show that tunicamycin and, more pronouncedly, a combination of tunicamycin and brefeldin A treatment caused misfolding of the luminal VP7 protein, leading to interdisulfide bond aggregation. While formation of VP7 aggregates could be prevented under reducing conditions, they reoccurred in less than 30 min after a shift to an oxidizing milieu. Furthermore, while glycosylated VP7 interacted during maturation with protein disulfide isomerase, nonglycosylated VP7 did not, suggesting that glycosylation is a prerequisite for protein disulfide isomerase interaction. While native NSP4, which does not possess S-S bonds, was not dependent on N-linked glycosylation or on protein disulfide isomerase assistance for maturation, nonglycosylated NSP4 was surprisingly found to interact with protein disulfide isomerase, further suggesting that protein disulfide isomerase can act both as an enzyme and as a chaperone. In conclusion, our data suggest that the major function of carbohydrates on VP7 is to facilitate correct disulfide bond formation and protein folding.
Transmission of Human T-Cell Leukemia Virus Type 1 to Mice
Authors: Fang Jianhua , Kushida Shigeki , Feng Renqing , Tanaka Masakazu , Kawamura Tomonori , Abe Hideaki , Maeda Naoyoshi , Onobori Makoto , Hori Mituo , Uchida Kazuhiko , Miwa Masanao 
Published: December 2019

ABSTRACT

Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and other diseases. For prevention of the transmission of HTLV-1 and manifestation of these diseases, a small-animal model, especially a mouse model, would be useful. We injected HTLV-1-producing T cells (MT-2) intraperitoneally into neonatal C3H/HeJ mice. While the antibody against HTLV-1 antigens was not detectable in C3H/HeJ mice, HTLV-1 provirus was frequently detected in the spleen, lymph nodes, and thymus by PCR. HTLV-1 provirus was present at the level of 0 to 30 molecules in 10
5
spleen cells at the age of 15 weeks. In addition, a 59-bp flanking sequence of the HTLV-1 integration site was amplified from the spleen DNA by linker-mediated PCR and was confirmed to be derived from the mouse genome. HTLV-1 provirus was found in the T-cell fraction of the mouse spleen. These results indicate that mice can be infected by HTLV-1 and could serve as an animal model for the study of HTLV-1 infection and its pathogenesis in vivo.
Cloning of Novel Isoforms of the Human Gli2 Oncogene and Their Activities To Enhance Tax-Dependent Transcription of the Human T-Cell Leukemia Virus Type 1 Genome
Authors: Tanimura Akira , Dan Shingo , Yoshida Mitsuaki 
Published: December 2019

ABSTRACT
The expression of human T-cell leukemia virus type 1 (HTLV-1) is activated by interaction of a viral transactivator protein, Tax, and cellular transcription factor, CREB (cyclic AMP response element binding protein), which bind to a 21-bp enhancer in the long terminal repeats (LTR). THP (Tax-helping protein) was previously determined to enhance the transactivation by Tax protein. Here we report novel forms of the human homolog of a member of the Gli oncogene family, Gli2 (also termed Gli2/THP), an extended form of a zinc finger protein, THP, which was described previously. Four possible isoforms (hGli2 α, β, γ, and δ) are formed by combinations of two independent alternative splicings, and all the isoforms could bind to a DNA motif, TRE2S, in the LTR. The longer isoforms, α and β, were abundantly expressed in various cell lines including HTLV-1-infected T-cell lines. Fusion proteins of the hGli2 isoforms with the DNA-binding domain of Gal4 activated transcription when the reporter contained a Gal4-binding site and one copy of the 21-bp sequence, to which CREB binds. This activation was observed only in the presence of Tax. The 21-bp sequence in the reporter was also essential for the activation. These results suggest that simultaneous binding of hGli2 and CREB to the respective sites in the reporter seems to be critical for Tax protein to activate transcription. Consequently, it is probable that the LTR can be regulated by two independent signals through hGli2 and CREB, since the LTR contains the 21-bp and TRE2S sequences in the vicinity.
Minimal Template Requirements for Initiation of Minus-Strand Synthesis In Vitro by the RNA-Dependent RNA Polymerase of Turnip Yellow Mosaic Virus
Authors: Deiman B. A. L. M. , Koenen A. K. , Verlaan P. W. G. , Pleij C. W. A. 
Published: December 2019

ABSTRACT

From mutational analysis of the 3′-terminal hairpin of turnip yellow mosaic virus (TYMV) RNA and use of nonstructured C-rich RNA templates, we conclude that the main determinant in the tRNA-like structure of TYMV RNA for initiation of minus-strand synthesis by the viral RNA-dependent RNA polymerase (RdRp) is the non-base-paired 3′ ACC(A) end. Base pairing of this 3′ end reduces the transcription efficiency drastically, and deletion of only the 3′-terminal A residue results in a fivefold drop in efficiency. The two C residues of the 3′ ACCA end are required for efficient transcription, as shown by substitution mutations. However, the 5′ A residue is not specifically involved in initiation of transcription, as shown by substitution mutations. Furthermore, the hairpin stem and loop upstream of the 3′ ACCA end also do not interact with the RdRp in a base-specific way. However, for efficient transcription, the hairpin stem should be at least five bp in length, while the calculated Δ
G
value should be less than −10.5 kcal/mol. Unexpectedly, the use of nonstructured C-rich RNA templates showed that the RdRp can start internally on an NCCN or NUCN sequence. Therefore, a possible function of the tRNA-like structure of TYMV RNA may be to prevent internal initiation of minus-strand synthesis.
The MouseH-2ARegion Influences the Envelope Gene Structure of Tumor-Associated Murine Leukemia Viruses
Authors: Nuckols J. Dean , Thomas Christopher Y. 
Published: December 2019

ABSTRACTC57BL/10 (B10) strains congenic at the mouse major histocompatibility locus (H-2) were injected with a modified ecotropic SL3-3 murine leukemia virus (MuLV) to determine the effect of theH-2genes on the envelope gene structure of recombinant MuLVs. All tested strains rapidly developed T-cell lymphomas, and recombinant proviruses were detected in the tumor DNAs by Southern blot. The B10.D2 (H-2d), B10.Br (H-2k), B10.Q (H-2q), and B10.RIII (H-2r) strains exhibited a TI phenotype in which almost all tumors contained type I recombinants. These recombinants characteristically acquire envelope gene sequences from the endogenous polytropic viruses but retain the 5′ p15E (TM) gene sequences from the ecotropic virus. The parental B10 (H-2b) strain, however, had a novel phenotype that was designated NS for nonselective. Only 30% of the B10 tumors had detectable type I recombinants, whereas a proportion of the others appeared to contain type II recombinants that lacked the type I-specific ecotropic p15E gene sequences. Studies of other B10 congenic strains with hybridH-2loci and selected F1animals revealed that the NS phenotype was regulated by a dominant gene(s) that mapped to the A region ofH-2b. These results demonstrate that a host gene within the major histocompatibility complex can influence the genetic evolution of pathogenic retroviruses in vivo.