Search results for "Recombinant DNA"
showing 4 items of 224 documents
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…
Single amino acid substitutions in the glycoprotein B carboxy terminus influence the fusion from without property of herpes simplex virus type 1.
1995
Syncytial mutations of herpes simplex virus type 1 (HSV-1) strains ANG, ANG path, HFEM, tsB5 and HSZP cause extensive cell fusion and were mapped to the cytoplasmic domain of glycoprotein B (gB), within the syn 3 locus. These strains are so far the only ones which show the phenotype ‘fusion from without’ (FFWO): 60 min after infection with high m.o.i., cells in a tissue culture are fused without transcription and translation of the viral genome. In this report we detected, using the recombinants 27/III and K-7, that an amino acid exchange from Ala to Val at aa position 854 of gB is the main determinant for FFWO activity of strains ANG, ANG path and recombinant K-7. The transfer of this muta…
Pseudovirions as Specific Tools for Investigation of Virus Interactions With Cells
2004
This chapter outlines the generation and application of human papillomavirus type 33 (HPV33) pseudovirions. The method describes (1) the construction of vaccinia viruses recombinant for the major and minor HPV capsid proteins, L1 and L2, respectively; (2) the transfection of Cos7 cells with a marker plasmid replicating to high copy numbers; (3) the expression of L1 and L2 using the vaccinia virus expression system; (4) the extraction, purification, and analysis of HPV33 pseudovirions; and (5) their use in pseudoinfection assays. These pseudovirions are structurally indistinguishable from native virions and are therefore valuable tools for the study of papillomavirus-cell interactions. The m…
Characterization of the autoantigen La (SS-B) as a dsRNA unwinding enzyme
1997
During the analysis of the La (SS-B) autoantigen for catalytic activities an ATP-dependent double-stranded RNA unwinding activity was detected. Both native and recombinant La proteins from different species displayed this activity, which could be inhibited by monospecific anti-La antibodies. La protein was able to melt dsRNA substrates with either two 3'-overhangs or a single 3'- and a 5'-overhang. Double-stranded RNAs with two 5'-overhangs were not unwound, indicating that at least one 3'-overhang is required for unwinding. Sequence elements of the La protein that might be involved in dsRNA unwinding, such as an evolutionarily conserved putative ATP-binding motif and an element that is hom…