Search

Binding of human immunodeficiency virus type 1 (HIV-1) to membrane of its target cells was studied by a quantitative and non-isotopic method called the viral membrane trapping method (VMTM). Membranes prepared from the CD4 positive lymphoblastoid cell line CEM and adsorbed to a solid support retained the ability to bind HIV-1. Similar results were obtained by Western dot blot and ELISA modification of VMTM, when membrane fraction was bound to nitrocellulose or polystyrene, respectively. In ELISA modification, viral association with 1 microgram of membranes coated on 96-well microplate was linear within a range of 3.75 micrograms to 60 micrograms of p24gag protein. The use of anti-CD4 mAbs, OKT4A and 13B8.2, identified CD4 molecule as a major HIV-1 binding component of membrane fraction. The procedure will allow the study of virus binding to this and to other possible additional receptor(s).

Membrane proteins (MP) obtained from the human mesenchymal rhabdomyosarcoma cell line RD were coated on 96-well polystyrene microplates and tested for their ability to bind human immunodeficiency virus type 1 (HIV-1). The virus bound to MP was detected by solid phase assay. Anti-human CD4 monoclonal antibodies directed against the HIV-1 gp120 binding site of the CD4 receptor did not inhibit viral binding to MP. HIV-1 specific polypeptides were recovered from coated MP to microplates by a modification of the solid phase immunoisolation technique and shown by immunoblotting analysis using a high titer of biotinylated human anti-HIV-1 IgG. Together these findings provide evidence that HIV-1 binding to RD cell surfaces can proceed via a mechanism other than those mediated by the CD4 receptor.

Simian syncitium-forming ("foamy") virus type I (SFV1) was characterized biochemically. RNA was extracted from purified virus either with 0.1 per cent SDS or by the standard phenol-chloroform method. By both techniques a main component of 65-70S was found. Denaturation of the 65-70S RNA by heat resulted in a shift of the sedimentation coefficient mainly to a 30-35S component. Electrophoresis on a composite polyacrylamide gel demonstrated the existence of three minor RNA's: 8S, 5S and 4S respectively. PAGE-SDS analysis of disrupted purified virions enabled the separate migration of five viral proteins and the identification of two main proteins: a 30 kd polypeptide and a 70 kd polypeptide.

Chromatography on heparin-Sepharose, known for its affinity for nucleotide-binding polypeptides, was used to purify the viral RNA-dependent DNA polymerase (reverse transcriptase) from the core polypeptides of simian foamy virus type 1. This procedure allowed the recovery of highly purified enzyme with a high specific activity. The average molecular weight of this monomeric enzyme is 81,000 and is thus comparable to that found for other known primate retroviruses. Reverse transcriptase activity of simian foamy virus type 1 requires a ribonucleotide template as a primer or otherwise a DNA with 3'-OH ends. Other optimal conditions of activity are reviewed. Heat inactivation studies led to the concept of an enzyme with two loci, one specific for the substrate and the other for the template-primer.

Simian foamy virus type 1 (SFV-1), the prototype of the Spumavirinae, was subjected to disruption and serial purification procedures. Separation of SFV-1 envelope components from viral cores was verified by electron microscopy, density gradient centrifugation and polyacrylamide gel electrophoresis. After affinity chromatography of the envelope polypeptides on a concanavalin A-Sepharose column, a highly purified 70 000 mol. wt. protein was recovered. Glycosylation of this gp70 was confirmed by glucosamine labelling. Immunological studies with anti-SFV antisera confirmed the type-specificity of this envelope gp70.

Spumavirinae or foamy viruses have been shown to have a characteristic RNA-dependent DNA polymerase activity. We demonstrate here the existence of an RNase H activity that copurifies with the 81-kilodalton monomeric polypeptide, which carries the RNA-dependent DNA polymerase activity of simian foamy virus type 1. RNase H degrades RNA hybrid substrates; however, it does not solubilize single-stranded RNAs. Inactivation assays with heat, high levels of bivalent cations, ethidium bromide, and sodium fluoride suggest that the RNase H catalytic site could be topologically independent from the DNA polymerase catalytic site.

The present study concerns the purification and partial characterization of simian foamy virus type 1 (SFV 1) structural core polypeptides. The obtention of SFV 1 cores separated from envelope components after viral disrupture was verified by electron microscopy (EM), density gradient, and polyacrylamide gel electrophoresis (PAGE). Multistep purification by column chromatography, verified by PAGE, enabled us to separate the structural core polypeptides from the 80,000 molecular weight reverse transcriptase. Two species of structural core polypeptides were identified with apparent molecular weights of 51 and 15 kd. By affinity chromatography on a double-stranded DNA-cellulose column, the main internal protein, p51, was shown to be composed of a major 30 kd protein and a minor 19 kd polypeptide, which binds to double stranded DNA. The p15 internal protein was shown to have a ribonucleotide binding nature.