CD4(81-92)-based peptide derivatives. Structural requirements for blockade of HIV infection, blockade of HIV-induced syncytium formation, and virostatic activity in vitro.

CD4(81-92) peptide block human immunodeficiency virus (HIV) infection, virus-induced cell fusion, and antigen production by HIV-1-infected cells when derivatized on specific amino acid residues. An extensive series of structural variants of 1,4,5-tribenzyl-10-acetyl-CD4(81-92) were tested as anti-viral agents in an attempt to define the sequence and derivatization requirements for antiviral activity, and to maximize potency and stability for use as potential therapeutic agents. Alteration of the primary amino acid sequence of the stem compound 1,4,5-tribenzyl-CD4(81-92) diminished or abolished in parallel all three indices of anti-viral activity in a series of altered sequence compounds. Replacement of d- for l-amino acid residues at positions 1, 2, 3, 4, 5, or 6 but not position 10 decreased anti-viral potency, again with parallel effects on infection, synctium formation, and virostatic activity. Omission of the glutamine residue at position 9 did not affect anti-viral potency, while removal of the glutamic acids at position 11 and 12 resulted in virtually complete loss of biological activity. Changes in the derivatization pattern of the CD4(81-92) peptide backbone also affected anti-viral potency and efficacy. Optimal activity was obtained with benzyl residues at positions 1, 4, and 5, whereas the 1,4,7-tribenzyl-CD4(81-92) compound was without activity in all assays tested. Replacement of one of the benzyl groups with an acetamidomethyl moiety resulted in complete loss of biological activity. The previously reported (Nara et al., Proc Natl Acad Sci USA 86: 7139-7143, 1989) virostatic activity of 1,4,5-tribenzyl-10-acetyl-CD4(81-92) (peptide #18) is apparently due to acetylation, since the desacetyl stem compound shows much less virostatic activity while still possessing full anti-infective and anti-syncytial activity, and acetylation of the N-terminus rather than the lysine of 1,4,5-tribenzyl-CD4(81-92) yields a virostatic compound equipotent to peptide #18. Cyclization of the tribenzyl peptide to further conformationally restrict the molecule resulted in a compound with anti-infection, anti-syncytial, and virostatic activity at submicromolar concentrations.

Molecular mass, biochemical composition, and physicochemical behavior of the infectious form of the scrapie precursor protein monomer.

A highly purified fraction obtained from scrapie (263-K strain)-infected hamsters' brains by an alternative procedure without proteinase K treatment contained a protease-resistant form of the scrapie precursor protein (PrPSc) and infectivity of 9.9 +/- 0.7 log LD50/ml. Polyclonal antibodies produced against hamster scrapie amyloid protein (PrP27-30) and used in a neutralization test diminished infectivity of the PrPSc preparations by 1.6 log after intracerebral inoculation and by 1 log after intraperitoneal inoculation. PrPSc was subjected to size-exclusion HPLC; greater than or equal to 60% of the eluted infectious units were recovered from the peak with an apparent mass of 30.4 +/- 0.6 kDa. Characterization by UV absorption spectra, SDS/PAGE, immunoblots, N-terminal amino acid sequence, and neutral sugar and amino sugar analyses demonstrated homogeneity of the infectious units. The neutral sugar and amino sugar compositional analyses revealed high mannose, glucosamine, fucose, and sialic acid content. This demonstrated an extensive posttranslational modification by the complex type of N-linked glycosylation and glycane core of C-terminal glycolipid of PrPSc. The results correspond to the predicted size, composition, and sequence of PrPSc and indicate that this protein may be the only component of scrapie infectious unit or the infectious form of scrapie precursor.

Large scale preparation of pure phycobiliproteins.

This paper describes simple procedures for the purification of large amounts of phycocyanin and allophycocyanin from the cyanobacterium Microcystis aeruginosa. A homogeneous natural bloom of this organism provided hundreds of kilograms of cells. Large samples of cells were broken by freezing and thawing. Repeated extraction of the broken cells with distilled water released phycocyanin first, then allophycocyanin, and provides supporting evidence for the current models of phycobilisome structure. The very low ionic strength of the aqueous extracts allowed allophycocyanin release in a particulate form so that this protein could be easily concentrated by centrifugation. Other proteins in the extract were enriched and concentrated by large scale membrane filtration. The biliproteins were purified to homogeneity by chromatography on DEAE cellulose. Purity was established by HPLC and by N-terminal amino acid sequence analysis. The proteins were examined for stability at various pHs and exposures to visible light.