Search results for "IDA"
showing 10 items of 33009 documents
Conformational Dynamics of the Dengue Virus Protease Revealed by Fluorescence Correlation and Single-Molecule FRET Studies.
2021
The dengue virus protease (DENV-PR) represents an attractive target for counteracting DENV infections. It is generally assumed that DENV-PR can exist in an open and a closed conformation and that active site directed ligands stabilize the closed state. While crystal structures of both the open and the closed conformation were successfully resolved, information about the prevalence of these conformations in solution remains elusive. Herein, we address the question of whether there is an equilibrium between different conformations in solution which can be influenced by addition of a competitive inhibitor. To this end, DENV-PR was statistically labeled by two dye molecules constituting a FRET …
Baculovirus display strategies: Emerging tools for eukaryotic libraries and gene delivery
2004
Recombinant baculoviruses have been extensively used as vectors for abundant expression of a large variety of foreign proteins in insect cell cultures. The appeal of the system lies essentially in easy cloning techniques and virus propagation combined with the eukaryotic post-translational modification machinery of the insect cell. Recently, a novel molecular biology tool was established by the development of baculovirus surface display, using different strategies for presentation of foreign peptides and proteins on the surface of budded virions. This eukaryotic display system enables presentation of large complex proteins on the surface of baculovirus particles and has thereby become a ver…
Single-chain antibodies against a plant viral RNA-dependent RNA polymerase confer virus resistance.
2004
Crop loss due to viral diseases is still a major problem for agriculture today. We present a strategy to achieve virus resistance based on the expression of single-chain Fv fragments (scFvs) against a conserved domain in a plant viral RNA-dependent RNA polymerase (RdRp), a key enzyme in virus replication. The selected scFvs inhibited complementary RNA synthesis of different plant virus RdRps in vitro and virus replication in planta. Moreover, the scFvs also bound to the RdRp of the distantly related hepatitis C virus. T(1) and T(2) progeny of transgenic lines of Nicotiana benthamiana expressing different scFvs either in the cytosol or in the endoplasmic reticulum showed varying degrees of r…
Biochemical and structural analysis of the NS5B RNA-dependent RNA polymerase of the hepatitis C virus.
2000
Hepatitis C virus (HCV), the major causative agent of chronic and sporadic non-A, non-B hepatitis worldwide, is a distinct member of the Flaviviridae virus family. These viruses have in common a plus-strand RNA genome that is replicated in the cytoplasm of the infected cell via minus-strand RNA intermediates. Owing to the lack of reliable cell culture systems and convenient animal models for HCV, the mechanisms governing RNA replication are not known. As a first step towards the development of appropriate in vitro systems, we expressed the NS5B RNA-dependent RNA polymerase (RdRp) in insect cells, purified the protein to near homogeneity and studied its biochemical properties. It is a primer…
Replication of herpes simplex virus type 1 and 2 in the medulla of the adrenal gland after vaginal infection of mice.
1996
After vaginal infections of mice with neuroinvasive strains of herpes simplex virus type 1 and 2 (HSV-1, HSV-2) virus replicates in the epithelium of the vagina, in the paravaginal ganglia, in the spinal cord and finally in the brain and in the adrenal glands. However, viral antigens could be demonstrated only in the medulla of the adrenal glands but not in the cortex, as assessed by immunohistochemistry (IHC). HSV could not be isolated from liver, spleen, uterus, and ovaries. This contrasts to the intraperitoneal (i.p) route of infection with replication in different visceral organs including the adrenal gland's cortex.
Binding and internalization of human papillomavirus type 33 virus-like particles by eukaryotic cells
1995
Infection of cells by human papillomaviruses (HPVs) associated with malignant genital lesions has not been studied because of the lack of an in vitro system and the unavailability of virions. We have now used virus-like particles (VLPs) of HPV type 33 to analyze the initial events in the interaction of the HPV capsid with cell lines. Binding of VLPs to HeLa cells was observed in biochemical assays and by immunofluorescence. VLP binding was inhibited by antisera raised against VLPs but not by monoclonal antibodies recognizing either L1 or L2 epitopes accessible on VLPs. Under saturating conditions, approximately 2 x 10(4) VLPs were bound per cell, with a dissociation constant of about 100 pM…
Assembly and Translocation of Papillomavirus Capsid Proteins
2002
ABSTRACT The major and minor capsid proteins of polyomavirus are preassembled in the cytoplasm and translocated to the nucleus only as a VP1-VP2/VP3 complex. In this study, we describe independent nuclear translocation of the L1 major protein and the L2 minor capsid protein of human papillomavirus type 33 by several approaches. First, we observed that expression and nuclear translocation of L2 in natural lesions precede expression of L1. Second, using a cell culture system for coexpression, we found that accumulation of L2 in nuclear domain 10 (ND10) subnuclear structures precedes L1 by several hours. In contrast, complexes of L2 and mutants of L1 forced to assemble in the cytoplasm are tra…
Nuclear localization but not PML protein is required for incorporation of the papillomavirus minor capsid protein L2 into virus-like particles.
2004
ABSTRACT Recent reports suggest that nuclear domain(s) 10 (ND10) is the site of papillomavirus morphogenesis. The viral genome replicates in or close to ND10. In addition, the minor capsid protein, L2, accumulates in these subnuclear structures and recruits the major capsid protein, L1. We have now used cell lines deficient for promyelocytic leukemia (PML) protein, the main structural component of ND10, to study the role of this nuclear protein for L2 incorporation into virus-like particles (VLPs). L2 expressed in PML protein knockout (PML −/− ) cells accumulated in nuclear dots, which resemble L2 aggregates forming at ND10 in PML protein-containing cells. These L2 assemblies also attracted…
Generation and neutralization of pseudovirions of human papillomavirus type 33
1997
Since human papillomaviruses (HPV) cannot be propagated in cell culture, the generation of infectious virions in vitro is a highly desirable goal. Here we report that pseudovirions can be generated by the assembly of virus-like particles (VLPs) in COS-7 cells containing multiple copies of a marker plasmid. Using recombinant vaccinia viruses, we have obtained spherical VLPs of HPV type 33 (HPV-33) which fractionate into heavy and light VLPs in cesium chloride density gradients. VLPs in the heavy fraction (1.31 g/cm3) carry the plasmid in DNase-resistant form and are capable of transferring the genetic marker located on the plasmid to COS-7 cells in a DNase-resistant way (pseudoinfection). Th…
Translocation of the nuclear autoantigen La to the cell surface of herpes simplex virus type 1 infected cells.
1992
Recently we developed a procedure to translocalize one of the extractable nuclear antigens (ENAs), the La protein, to the cell surface of CV-1 cells. Here we report that herpes simplex virus type 1 infection can also induce a translocation of the autoantigen to the cell surface. On the cell surface we detected La protein assembled with large protrusions. Within these protrusions La protein colocalized with virus particles. These protrusions are known to be released from the cell after virus infections. Such complexes consisting of self and virus could provide helper determinants for an anti-self response, and therefore be important in generation of autoimmunity.