Processing of in vitro-synthesized gag precursor proteins of human immunodeficiency virus (HIV) type 1 by HIV proteinase generated in Escherichia coli

Abstract

We expressed the gag and proteinase regions of human immunodeficiency virus (HIV) type 1 by transcription and translation in vitro. A synthetic RNA spanning the gag and pro domains gave primarily the unprocessed capsid precursor pr53. Efficient cleavage of this precursor was observed when the gag and pro domains were placed in the same translational reading frame, yielding equimolar amounts of the gag protein and of proteinase (PR). Expression of HIV type 1 PR in Escherichia coli as a fusion protein gave rapid autocatalytic processing to an HIV-specific protein of approximately 11 kilodaltons. HIV PR generated in E. coli specifically induced cleavage of the HIV capsid precursor, whereas deletion of the carboxy-terminal 17 amino acids of the proteinase rendered it inactive. Inhibitor studies showed that the enzyme was insensitive to inhibitors of serine and cysteine proteinases and metalloproteinases and was inhibited only by a very high concentration (1 mM) of pepstatin A.

In Vitro Assembly Properties of Purified Bacterially Expressed Capsid Proteins of Human Immunodeficiency Virus

“…Furthermore, efficient in vitro assembly in this case was dependent on added RNA. Identical structures were formed upon addition of either an HIV-derived RNA containing the putative viral packaging signal [44] or of total E. coli RNA. Incubation of CA-NC in pH 8, 0.1 M salt buffer at a concentration of 0.2 mg/ml (= 6 pM) and in the presence of added RNA (6 yg/ml) yielded approximately the same amount of structures as observed for CA at a protein concentration of 2 mg/ml and in high salt (Table 1).…”

Domains upstream of the protease (PR) in human immunodeficiency virus type 1 Gag-Pol influence PR autoprocessing

“…Since each Gag-Pol precursor contains only one PR domain, Gag-Pol precursor dimerization is necessary for the formation of the PR active site. However, additional mechanisms of regulation must be present during assembly and budding because HIV Gag-Pol precursors can undergo autoprocessing in the cytoplasm of some cells and in some in vitro systems (14,16,28). Moreover, in type C viruses, PR activation does not occur before the viral particle is completely released from the host cell even though viral precursors are highly concentrated at this stage (33).…”

“…To study only the effects of upstream sequences on PR activity, a stop codon was placed at the C terminus of the PR-coding region. The exclusive synthesis of PR-containing precursors was ensured by introducing 4 bp next to the natural frameshift site (16). Plasmids were grown in Escherichia coli C600 and JM101, and DNA was prepared by using the Promega Magic Mini Prep system.…”

Recombinant protein production driven by the tryptophan promoter is tightly controlled in ICONE 200, a new genetically engineeredE. coli mutant

“…The poliovirus 2B gene was amplified by PCR with the plasmid pT7.2B (Lama and Carrasco, 1992) as template and the primers PO2.1: 5Ј-GAATTCTGGCATCACCAAT-TACATAG-3Ј and PO2.2: 5Ј-AAGCTTAGTGGTGGTG-GTGGTGGTGTTGCTTGATGACATAAGGTATC-3Ј. A DNA sequence from the HIV-type 1 pol gene was amplified with the primers PR1: 5Ј-GCGAATTCTGATCTGGCCT-TCCTACAAGGGAAG-3Ј and PR2: 5Ј-CGAAGCTTTTA-AAAATTTAAAGTGCAACCAATCTG-3Ј and the plas-mid pHIV-ProPII (Kräusslich et al, 1988) as template. This sequence encoded a precursor of 151 residues containing the 99 amino acids of the protease.…”