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At least 217 records · Page 12Linked to original sources
PubMed2003

The human herpesvirus 6 U100 gene product is the third component of the gH-gL glycoprotein complex on the viral envelope.

The human herpesvirus 6 (HHV-6) variant A U100 gene encodes the third component of the glycoprotein H (gH)-glycoprotein L (gL)-containing complex. Glycosidase digestion analysis showed that the U100 gene products are glycoproteins consisting of an 80-kDa protein with complex N-linked oligosaccharides and a 74-kDa protein with immature, high-mannose N-linked oligosaccharides. Based on these characteristics, we designated the U100 gene products glycoprotein Q (gQ). Only the 80-kDa form of gQ was coimmunoprecipitated with an anti-gH antibody, suggesting that the 80-kDa protein associates with the gH-gL complex in HHV-6-infected cells. Furthermore, the complex was detected in purified virions, suggesting that it may play an important role in viral entry.

Cells, Cultured↗
PubMed1998

Major histocompatibility complex class I viral antigen processing in the secretory pathway defined by the trans-Golgi network protease furin.

Classical antigen presentation by major histocompatibility complex class I molecules involves cytosolic processing of endogenously synthesized antigens by proteasomes and translocation of processed peptides into the endoplasmic reticulum (ER) by transporters associated with antigen presentation (TAP). Alternative pathways for processing of endogenous antigens, generally involving the ER, have been suggested but not fully proved. We analyzed the potential for class I presentation of proteolytic maturation of secretory antigens in the exocytic pathway. We found that hepatitis B (HB) virus secretory core protein HBe can efficiently deliver COOH-terminally located antigenic peptides for endogenous class I loading in the absence of TAP. Antigen presentation to specific cytotoxic T lymphocytes correlates with protein maturation at the COOH terminus, since modification of maturation and transport of HBe through the secretory pathway alters antigen presentation. Both maturation and a necessary processing step occur in the Golgi or post-Golgi compartment. Antigen presentation is independent of proteasome activity, but inhibitors of the trans-Golgi network resident protease furin inhibit both HBe maturation and antigen presentation. These results define a new antigen processing pathway located in the secretory route, with a central role for proteolytic maturation mediated by the subtilisin protease family member furin as an efficient source for antigen presentation.

ATP-Binding Cassette Transporters↗
PubMed1972

State of adenovirus 2 deoxyribonucleic acid in the nucleus and its mode of transcription: studies with isolated viral deoxyribonucleic acid-protein complexes and isolated nuclei.

Newly replicated adenovirus 2 deoxyribonucleic acid (DNA) can be isolated from the nucleus of HeLa cells by a gentle lysis procedure as a fairly homogeneous complex with a sedimentation of 73S. The viral DNA complex can be prepared completely free from host cell DNA. The viral complex is slightly active in ribonucleic acid (RNA) synthesis in vitro. Treatment of the complex with Pronase and sodium dodecyl sulfate converts the DNA to a form which sediments at 43S. Nuclei isolated from adeno-infected cells synthesize high-molecular-weight virus-specific RNA in vitro. Optimal RNA synthesis requires a divalent cation, preferentially manganese, and relatively high salt concentrations. The synthesis of virus-specific RNA by the isolated nuclei is strongly inhibited by low doses of alpha-amanitine. The latter experimental result is discussed in terms of the polymerase used to transcribe the adenovirus DNA in vivo.

Adenoviridae↗
PubMed2006

Evolution of complexity in the viral world: the dawn of a new vision.

Recent sequencing of the genomes of numerous large viruses provide for unprecedented opportunities to study the emergence and evolution of complexity in the virus world. This special issue of Virus Research explores trends in the evolution of complex genomes in most major classes of viruses.

Evolution, Molecular↗
PubMed1988

Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI.

Evidence was recently presented that herpes simplex virus type 1 (HSV-1) immunoglobulin G (IgG) Fc receptors are composed of a complex containing a previously described glycoprotein, gE, and a novel virus-induced polypeptide, provisionally named g70 (D. C. Johnson and V. Feenstra, J. Virol. 61:2208-2216, 1987). Using a monoclonal antibody designated 3104, which recognizes g70, in conjunction with antipeptide sera and virus mutants unable to express g70 or gE, we have mapped the gene encoding g70 to the US7 open reading frame of HSV-1 adjacent to the gE gene. Therefore, g70 appears to be identical to a recently described polypeptide which was named gI (R. Longnecker, S. Chatterjee, R. J. Whitley, and B. Roizman, Proc. Natl. Acad. Sci. USA 84:147-151, 1987). Under mildly denaturing conditions, monoclonal antibody 3104 precipitated both gI and gE from extracts of HSV-1-infected cells. In addition, rabbit IgG precipitated the gE-gI complex from extracts of cells transfected with a fragment of HSV-1 DNA containing the gI, gE, and US9 genes. Cells infected with mutant viruses which were unable to express gE or gI did not bind radiolabeled IgG; however, cells coinfected with two viruses, one unable to express gE and the other unable to express gI, bound levels of IgG approaching those observed with wild-type viruses. These results further support the hypothesis that gE and gI form a complex which binds IgG by the Fc domain and that neither polypeptide alone can bind IgG.

Animals↗
PubMed1994

Mapping of a region of the paramyxovirus L protein required for the formation of a stable complex with the viral phosphoprotein P.

The paramyxovirus large protein (L) and phosphoprotein (P) are both required for viral RNA-dependent RNA polymerase activity. Previous biochemical experiments have shown that L and P can form a complex when expressed from cDNA plasmids in vivo. In this report, L and P proteins of the paramyxovirus simian virus 5 (SV5) were coexpressed in HeLa T4 cells from cDNA plasmids, and L-P complexes were examined. To identify regions of the SV5 L protein that are required for L-P complex formation, 16 deletion mutants were constructed by mutagenesis of an SV5 L cDNA. Following coexpression of these L mutants with cDNA-derived P and radiolabeling with 35S-amino acids, cell lysates were analyzed for stable L-P complexes by a coimmunoprecipitation assay and by sedimentation on 5 to 20% glycerol gradients. Mutant forms of L containing deletions that removed as much as 1,008 residues from the C-terminal half of the full-length 2,255-residue L protein were detected in complexes with P by these two assays. In contrast, large deletions in the N-terminal half of L resulted in proteins that were defective in the formation of stable L-P complexes. Likewise, L mutants containing smaller deletions that individually removed N-terminal regions which are conserved among paramyxovirus and rhabdovirus L proteins (domain I, II, or III) were also defective in stable interactions with P. These results suggest that the N-terminal half of the L protein contains sequences important for stable L-P complex formation and that the C-terminal half of L is not directly involved in these interactions. SV5-infected HeLa T4 cells were pulse-labeled with 35S-amino acids, and cell extracts were examined by gradient sedimentation. Solubilized L protein was detected as an approximately 8 to 10S species, while the P protein was found as both a approximately 4S form (approximately 85%) and a species that cosedimented with L (approximately 15%). These data provide the first biochemical evidence in support of a simple domain structure for an L protein of the nonsegmented negative-sense RNA viruses. The results are discussed in terms of a structural model for the L protein and the interactions of L with the second viral polymerase subunit P.

Base Sequence↗
PubMed2006

Phosphorylation of human respiratory syncytial virus P protein at threonine 108 controls its interaction with the M2-1 protein in the viral RNA polymerase complex.

The human respiratory syncytial virus (HRSV) P protein is phosphorylated, with different turnover rates, at several serine (S) and threonine (T) residues. The role of phosphothreonines in viral RNA synthesis was studied by using P protein substitution variants and the HRSV-based minigenome pM/SH. By using liquid chromatography coupled to ion-trap mass spectrometry, it was found that P protein T108 was phosphorylated by addition of a high-turnover phosphate group. This phosphorylation occurs in P protein expressed transiently and during HRSV infection. The results suggest that phosphorylation at P protein T108 affects M2-1 transcriptional activities, because this modification prevents interaction between the P and M2-1 proteins. Therefore, P protein phosphorylation-dephosphorylation at T108 could distinguish the role of the P protein in viral transcription and replication.

Amino Acid Substitution↗
PubMed1984

Protein composition of adenovirus nucleoprotein complexes extracted from infected cells.

A viral nucleoprotein complex was extracted from the nuclei of human cells 20 hr after infection with adenovirus type 2 or several of its temperature-sensitive mutants. In its sedimentation property, density in CsCl, and digestion pattern with micrococcal nuclease, the complex resembled viral cores. The polypeptides V, PVII, 11K, and 36K were found associated with this complex which is formed prior to or in the absence of virus assembly. The results suggest that this nucleoprotein complex is a direct precursor to virus assembly.

Adenoviruses, Human↗
PubMed1998

Modulation of natural killer cell cytotoxicity in human cytomegalovirus infection: the role of endogenous class I major histocompatibility complex and a viral class I homolog.

Natural killer (NK) cells have been implicated in early immune responses against certain viruses, including cytomegalovirus (CMV). CMV causes downregulation of class I major histocompatibility complex (MHC) expression in infected cells; however, it has been proposed that a class I MHC homolog encoded by CMV, UL18, may act as a surrogate ligand to prevent NK cell lysis of CMV-infected cells. In this study, we examined the role of UL18 in NK cell recognition and lysis using fibroblasts infected with either wild-type or UL18 knockout CMV virus, and by using cell lines transfected with the UL18 gene. In both systems, the expression of UL18 resulted in the enhanced killing of target cells. We also show that the enhanced killing is due to both UL18-dependent and -independent mechanisms, and that the killer cell inhibitory receptors (KIRs) and CD94/NKG2A inhibitory receptors for MHC class I do not play a role in affecting susceptibility of CMV-infected fibroblasts to NK cell-mediated cytotoxicity.

Antigens, Viral↗
PubMed1991

Formation and intracellular transport of a heterodimeric viral spike protein complex.

We have analyzed the heterodimerization and intracellular transport from the ER to the Golgi complex (GC) of two membrane glycoproteins of a bunyavirus (Uukuniemi virus) that matures by a budding process in the GC. The glycoproteins G1 and G2, which form the viral spikes, are cotranslationally cleaved in the ER from a 110,000-D precursor. Newly synthesized G1 was transported to the GC and incorporated into virus particles about 30-45 min faster than newly synthesized G2. Analysis of the kinetics of intrachain disulfide bond formation showed that G1 acquired its mature form within 10 min, while completion of disulfide bond formation of G2 required a considerably longer time (up to 60 min). During the maturation process, G2 was transiently associated with the IgG heavy chain binding protein for a longer time than G1. Protein disulfide isomerase also coprecipitated with antibodies against G1 and G2. In virus particles, G1 and G2 were present exclusively as heterodimers. Immunoprecipitation with monoclonal antibodies showed that heterodimerization occurred rapidly, probably in the ER, between newly made G1 and mature, dimerization competent G2. Taken together, our results show that these two viral glycoproteins have different maturation kinetics in the ER. We conclude that the apparent different kinetics of ER to GC transport of G1 and G2 is due to the different rates by which these proteins fold and become competent to enter into heterodimeric complexes prior to exit from the ER.

Animals↗
PubMed1992

Molecular modeling of an antigenic complex between a viral peptide and a class I major histocompatibility glycoprotein.

Computer simulation of the conformations of short antigenic peptides (5-10 residues) either free or bound to their receptor, the major histocompatibility complex (MHC)-encoded glycoprotein H-2 Ld, was employed to explain experimentally determined differences in the antigenic activities within a set of related peptides. Starting for each sequence from the most probable conformations disclosed by a pattern-recognition technique, several energy-minimized structures were subjected to molecular dynamics simulations (MD) either in vacuo or solvated by water molecules. Notably, antigenic potencies were found to correlate to the peptides propensity to form and maintain an overall alpha-helical conformation through regular i,i + 4 hydrogen bonds. Accordingly, less active or inactive peptides showed a strong tendency to form i,i + 3 hydrogen bonds at their N-terminal end. Experimental data documented that the C-terminal residue is critical for interaction of the peptide with H-2 Ld. This finding could be satisfactorily explained by a 3-D Q.S.A.R. analysis postulating interactions between ligand and receptor by hydrophobic forces. A 3-D model is proposed for the complex between a high-affinity nonapeptide and the H-2 Ld receptor. First, the H-2 Ld molecule was built from X-ray coordinates of two homologous proteins: HLA-A2 and HLA-Aw68, energy-minimized and studied by MD simulations. With HLA-A2 as template, the only realistic simulation was achieved for a solvated model with minor deviations of the MD mean structure from the X-ray conformation. Water simulation of the H-2 Ld protein in complex with the antigenic nonapeptide was then achieved with the template-derived optimal parameters. The bound peptide retains mainly its alpha-helical conformation and binds to hydrophobic residues of H-2 Ld that correspond to highly polymorphic positions of MHC proteins. The orientation of the nonapeptide in the binding cleft is in accordance with the experimentally determined distribution of its MHC receptor-binding residues (agretope residues). Thus, computer simulation was successfully employed to explain functional data and predicts alpha-helical conformation for the bound peptide.

Computer Simulation↗
PubMed2003

Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA.

Biochemical studies revealed that nonstructural proteins of hepatitis C virus (HCV) interacted with each other and were associated with intracellular membranes. The goals of this study were to determine whether nonstructural viral proteins are colocalized at specific intracellular sites where HCV RNA is replicated and to identify the virus components of the HCV replication complex (RC). Immunofluorescence and subcellular fractionation studies were performed to determine the intracellular colocalization of nonstructural HCV proteins and the replicating RNA in a human hepatoma cell line, Huh7, in which a subgenomic HCV RNA was replicated persistently. The replicating HCV RNA was labelled with 5-bromouridine 5'-triphosphate (BrUTP). Results show that each of the nonstructural HCV proteins was colocalized predominantly with the newly synthesized HCV RNA labelled with BrUTP and an endoplasmic reticulum (ER) protein, calnexin. Consistent with these findings, subcellular fractionation and Western blot analyses revealed that the nonstructural HCV proteins were colocalized with HCV RNA mainly in the membrane fractions. Conversely, the viral nonstructural proteins and RNA remained in the soluble fractions upon treatment with detergent, confirming the membrane association of the HCV RC. HCV RNA in the membrane-bound RC was resistant to RNase treatment, whereas it became sensitive to RNases once the membranes were disrupted by treatment with detergent, suggesting that the HCV RC is assembled within membrane structures. Collectively, these findings demonstrate that HCV RNA replication occurs in the perinuclear ER membrane-bound HCV RC, containing nonstructural viral proteins and RNA.

Cell Membrane↗
PubMed2006

Assembly of an active translation initiation factor complex by a viral protein.

Recruitment of the 40S ribosome to the 5' end of a eukaryotic mRNA requires assembly of translation initiation factors eIF4E, the cap-binding protein, together with eIF4A and eIF4G into a complex termed eIF4F. While the translational repressor 4E-BP1 regulates binding of eIF4E to eIF4G, the forces required to construct an eIF4F complex remain unidentified. Here, we establish that the herpes simplex virus-1 (HSV-1) ICP6 polypeptide associates with eIF4G to promote eIF4F complex assembly. Strikingly, release of eIF4E from the 4E-BP1 repressor is insufficient to drive complex formation, suggesting that ICP6 is an eIF4F-assembly chaperone. This is the first example of a translation initiation factor-associated protein that promotes active complex assembly and defines a new, controllable step in the initiation of translation. Homology of the N-terminal, eIF4G-binding segment of ICP6 with cellular chaperones suggest that factors capable of interacting with eIF4G and promoting eIF4F complex assembly may play important roles in a variety of processes where translation complexes need to be remodeled or assembled on populations of newly synthesized or derepressed mRNAs, including development, differentiation, and the response to a broad spectrum of environmental cues.

Cell Line↗
PubMed2001

Association of immune complexes and plasma viral load with CD4+ cell depletion, CD8+ DR+ and CD16+ cell counts in HIV+ hemophilia patients. Implications for the immunopathogenesis of HIV-induced CD4+ lymphocyte depletion.

OBJECTIVE: There is evidence that HIV induces CD4+ depletion in part by the formation of immune complexes (IC) that attach to CD4+ blood lymphocytes. In the present study we examined the relationship of IC-coated CD4+ blood cells with retroviral replication in HAART-treated patients. PATIENTS AND METHODS: 52 hemophilia patients were studied from 1997 to 1999. Lymphocyte subsets, IgM, IgG and gp120 on CD4+ blood cells, in vitro responses of lymphocytes to mitogens, plasma neopterin and plasma viral load were measured. RESULTS: Patients with detectable viral replication and without ICs on CD4+ blood lymphocytes had a lower viral load (4100 versus 21000 HIV-1 mRNA copies/ml; P = 0.079) and higher CD4+ cell counts (310/microl versus 161/microl; P = 0.035) than patients with ICs on circulating CD4+ lymphocytes. Among patients with < 80 HIV-1 mRNA copies/ml, IC- individuals had slightly higher CD4+ lymphocyte counts than IC+ patients (384/microl versus 316/microl; n.s.). Further evidence for the clinical relevance of the ICs was obtained when 18 patients who had an undetectable viral load at previous investigations were analyzed. Among patients with a stable undetectable viral load, CD4+ counts increased in 6 of 8 IC- but in none of 2 IC+ individuals. In patients whose viral load increased during the observation period, 5 of 6 IC- but none of 2 IC+ individuals showed higher CD4+ cell counts. Impaired virus killing is suggested by lower CD16+ (35/microl versus 107/microl; P = 0.016), higher CD3+ DR+ (178/microl versus 66/microl; P = 0.006), and higher CD8+ DR+ (142/microl versus 34/microl; P = 0.017) cell counts in IC(-) patients compared to IC- patients without detectable viral load. Strong retroviral replication induced strong T cell dysfunctions. Fewer CD3+ 25+ blood lymphocytes (19/microl versus 47/microl; P = 0.006) and a lower in vitro response of T lymphocytes to the mitogens Con A (RR: 0.3 versus 1.2; P=0.023) and CD3 mab (RR: 0.5 versus 2.4; P = 0.012) was observed in IC+ patients with detectable versus undetectable viral load. CONCLUSION: Our data suggest that ICs on circulating CD4+ blood lymphocytes are primarily associated with CD4+ lymphocyte depletion whereas the plasma viral load is primarily associated with decreased T lymphocyte activation, lower CD16+ counts, and higher CD8+ DR+ lymphocytes which might be the effector cells for virus elimination.

Antigen-Antibody Complex↗
PubMed1997

RNA-dependent RNA polymerase activity associated with the yeast viral p91/20S RNA ribonucleoprotein complex.

20S RNA is a noninfectious viral single-stranded RNA found in most laboratory strains of the yeast Saccharomyces cerevisiae. 20S RNA encodes a protein of 91 kDa (p91) that contains the common motifs found among RNA-dependent RNA polymerases from RNA viruses. p91 and 20S RNA are noncovalently associated in vivo, forming a ribonucleoprotein complex. We detected an RNA polymerase activity in p91/20S RNA complexes isolated by high-speed centrifugation. The activity was not inhibited by actinomycin D nor alpha-amanitin. The majority of the in vitro products was 20S RNA and the rest was the complementary strands of 20S RNA. Because the extracts were prepared from cells accumulating 20S RNA over its complementary strands, these in vitro products reflect the corresponding activities in vivo. When the p91/20S RNA complexes were subjected to sucrose gradient centrifugation, the polymerase activity cosedimented with the complexes. Furthermore, an RNA polymerase activity was detected in the complex by an antibody-linked polymerase assay using anti-p91 antiserum, suggesting that p91 is present in the active RNA polymerase machinery. These results together indicate that p91 is the RNA-dependent RNA polymerase or a subunit thereof responsible for 20S RNA replication.

Amanitins↗