Determinants of the human immunodeficiency virus type 1 p15NC-RNA interaction that affect enhanced cleavage by the viral protease.

During human immunodeficiency virus type 1 (HIV-1) virion assembly, cleavage of the Gag precursor by the viral protease results in the transient appearance of a nucleocapsid-p1-p6 intermediate product designated p15NC. Utilizing the p15NC precursor protein produced with an in vitro transcription-translation system or purified after expression in Escherichia coli, we have demonstrated that RNA is required for efficient cleavage of HIV p15NC. Gel mobility shift and nitrocellulose filter binding experiments indicate that purified p15NC protein specifically binds its corresponding mRNA with an estimated Kd of 1.5 nM. Binding was not affected by the presence or absence of zinc or EDTA. Moreover, mutagenesis of the cysteine residues within either of the two Cys-His arrays had no effect on RNA binding or on RNA-dependent cleavage by the viral protease. In contrast, decreased binding of RNA and diminished susceptibility to cleavage in vitro were observed with p15NC-containing mutations in one or more residues within the triplet of basic amino acids present in the region between the two zinc fingers. In addition, we found that 21- to 24-base DNA and RNA oligonucleotides of a particular sequence and secondary structure could substitute for p15 RNA in the enhancement of p15NC cleavage. Virus particles carrying a mutation in the triplet of NC basic residues (P3BE) show delayed cleavage of p15NC and a defect in core formation despite the eventual appearance of fully processed virion protein. These results define determinants of the p15NC-RNA interaction that lead to enhanced protease-mediated cleavage and demonstrate the importance of the triplet of basic residues in formation of the virus core.

Construction of infectious molecular clones of HIV-1 containing defined mutations in the protease gene.

A DNA clone of HIV-1 containing the full-length infectious viral sequence was cleaved at a unique Nco I restriction site within the viral genome, and DNA fragments containing the 5' and 3' portions of the HIV genome were subcloned into separate plasmid vectors. The 5' 'half-virus' construct was further modified by incorporating a class IIS restriction site, Esp3I, near the 3' end of the protease gene of HIV. This site, in combination with a natural ApaI site near the 5' end of the protease gene, creates a convenient cassette shuttle vector in which the protease coding region can be easily replaced. Recombinant viruses containing protease genes either altered by site-directed mutagenesis or amplified from clinical or laboratory isolates can be reconstructed. The DNA fragment containing the protease gene is first subcloned into the 5' half-virus shuttle vector plasmid. Infectious recombinant virus is subsequently recovered by cotransfecting 5' and 3' half-virus plasmids linearized at their common Nco I sites into mammalian cells. This method was successfully applied to constructing viruses containing various substitutions in protease.

Expression and functional characterization of recombinant human vascular cell adhesion molecule-1 (VCAM1) synthesized by baculovirus-infected insect cells.

Vascular cell adhesion molecule-1 (VCAM1) is a cell surface glycoprotein produced by the vascular endothelium, as well as on macrophage-like and dendritic cell types, in response to certain inflammatory stimuli. VCAM1 interacts with the integrin VLA4 present on mononuclear leukocytes. We have isolated the cDNA for VCAM1 using RT-PCR by screening a cDNA library from IL-1 beta-activated human endothelial cells. To obtain large quantities of VCAM1 for structural and functional studies, we have produced this protein in insect cells using a baculovirus expression system. Insect cells infected with recombinant virus synthesized human VCAM1 at levels exceeding 3% of total cellular protein following 72 h postinfection. VCAM1-expressing insect cells were shown to bind specifically to a variety of VLA4 expressing cell lines (Jurkat, THP-1, U937). Thus, recombinant VCAM1 protein produced in the baculovirus expression system was localized to the cell surface and was biologically active. Large-scale availability of this adhesion protein should enhance efforts toward the discovery of new antiadhesive (anti-inflammatory and antiatherogenic) therapeutics.

Permuteins of interleukin 1 beta--a simplified approach for the construction of permutated proteins having new termini.

A technique for the rapid and simple generation of permutated versions of the interleukin-1 beta (IL-1 beta) gene is described. In this method, the human IL-1 beta cDNA is twice amplified by the polymerase chain reaction (PCR) and the resulting DNA fragments are ligated in tandem. Between the two genes, the DNA sequence encodes a short four amino acid loop to link the native N- and C-terminal ends of the IL-1 beta protein. By using PCR amplification from this starting template, a new version of the IL-1 beta cDNA was obtained that encodes a permutated form of the IL-1 beta protein where the new N- and C-terminal amino acids correspond to residues 65 and 64 of the native IL-1 beta sequence, respectively. The name 'permutein' is proposed to describe proteins generated by this technology. The molecular profile (IL-1 receptor binding, biologic activity and solution properties) of the IL-1 permutein produced by this technology, permutein 65/64, is shown to be identical to that of native IL-1 beta. The approach should be useful to define further the structural features of this protein that are important for its function.

Mutagenesis of protease cleavage sites in the human immunodeficiency virus type 1 gag polyprotein.

The virally encoded protease of human immunodeficiency virus (HIV) is responsible for specific cleavage events leading to the liberation of the enzymes reverse transcriptase, integrase, ribonuclease H, and the core proteins from the gag-pol and gag polyprotein precursors. Utilizing gag polyprotein synthesized in vitro, we have shown that this substrate is sequentially cleaved by purified HIV protease to yield products that on the basis of their sizes and immunoreactivities correspond to p15, p6, p7, p17, and finally mature p24. We have placed unique restriction sites flanking the p17-p24 domain in order to facilitate replacement of cleavage site sequences by utilizing oligonucleotide cassettes. Replacement of the rapidly cleaved methionine-methionine bond at the p24-p15 junction with tyrosine-proline or replacement of the tyrosine-proline bond at the p17-p24 junction with methionine-methionine results in sites that cannot be efficiently cleaved. A basic amino acid at the p17-p24 scissile bond is not tolerated. Replacement of this cleavage site with an inverted repeat amino acid sequence gives intermediate rates of cleavage. In an attempt to convert the p17-p24 domain into a p24-p15 domain, residues flanking the scissile bond were exchanged in an expanding iterative fashion. When four residues flanking the scissile bond had been replaced, the rate of cleavage relative to that of the native p17-p24 sequence was increased fourfold. The cleavage rate of the native p24-p15 sequence is still some 10-fold greater than that of the p17-p24 sequence, suggesting that more-distant residues significantly affect the cleavage rate.