Bovine leukemia virus protease: purification, chemical analysis, and in vitro processing of gag precursor polyproteins

Abstract

Bovine leukemia virus protease was purified to homogeneity and assayed by using murine leukemia virus Pr65gag, a polyprotein precursor of the viral core structural proteins, as the substrate. A chemical analysis of the protease, including an amino acid composition and NH2-and COOH-terminal amino acid sequence analysis, revealed that it has an Mr of 14,000 and is encoded by a segment of the viral RNA located between the gag gene and the putative reverse transcriptase gene. As expected from the nucleotide sequence data (Rice et al., Virology 142:357-377, 1985), the reading frame for the protease is different from both the gag and reverse transcriptase reading frames. The 5' end of the protease open reading frame extends 38 codons upstream from the codon for the NH2-terminal residue of the mature viral protease and overlaps the gag open reading frame by 7 codons. The 3' end of the protease open reading frame extends 26 codons beyond the codon for the COOH-terminal residue of the mature protease and overlaps 8 codons of the reverse transcriptase open reading frame. Several lines of evidence, such as protein mapping of the gag polyprotein precursor, the characteristic structure of the mRNA, and promotion of the synthesis of a gag polyprotein precursor by lysine tRNA in vitro, suggest that the protease could be translated by frameshift suppression of the gag termination codon. In vitro synthesized bovine leukemia virus gag-related polyproteins were cleaved by the protease into fragments which were the same size as the known components of bovine leukemia virus, suggesting that the specificity of cleavage catalyzed in vitro by the purified protease is the same as the specificity of cleavage found in the virus.

A Nucleotide Deletion Causing a Translational Stop in the Protease Reading Frame of Bovine Leukaemia Virus (BLV) Results in Modified Protein Expression and Loss of Infectivity

“…1. The amino‐and carboxy‐terminal sequences of purified BLV protease agree with the amino acid sequence deduced from the nucleotide sequence from positions 1680 to 1764 and from positions 2052 to 2058 in Fig. 1 ( Yoshinaka et al, 1986; Menard et al, 1993). If the frameshifts do not occur, translation will be terminated by stop codons downstream from the frameshift site.…”

“…The new isolates allow the translation of the gag‐prt‐pol region in the same way as the Belgian isolate ( Fig. 1), taking into account the frameshift sites described by Hatfield et al (1989) and the partial amino acid sequences of the BLV protease ( Yoshinaka et al, 1986). Only the Australian and the Japanese BLV‐tumour provirus contain a deletion of one nucleotide in the prt frame followed by a ‐ 1 frameshift at an incorrect position.…”

Bovine leukemia virus matrix-associated protein MA(p15): further processing and formation of a specific complex with the dimer of the 5'-terminal genomic RNA fragment

“…Organization of the BLV gag precursor protein. As pre-sented in earlier publications by us (42) and others (40), BLV particles prepared from FLK and Bat2Cll cell cultures have as a major component a 10-kDa protein, pi0, as well as MA(pl5), CA(p24), NC(p12), and PR(p14). Although plO can be separated by a standard RP-HPLC procedure from other viral proteins (42), neither its origin nor its function has been described.…”

“…The BLV gag proteins MA(plS), p10, and NC(p12) and the PR were isolated by fractionation of a 1.0 M NaCl extract of BLV acetone powder on a Sephacryl S-200 (Pharmacia) column, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) (42). RP-HPLC conditions and the separation profiles of NC(p12), MA(plO), and PR are given in reference 42. MA(pl5) was purified by the same procedure from different fractions of an earlier eluate in the Sephacryl S-200 column chromatography.…”

Processing of gag precursor polyprotein of human T-cell leukemia virus type I by virus-encoded protease

“…Proteolytic cleavage of the gag precursor polyprotein of murine and avian leukemogenic retroviruses has been shown to be carried out by a highly substrate-specific virionassociated protease, which plays an important role in infectivity of virus particles (17,48). On the basis of comparison of the amino acid sequence of retrovirus-associated proteases with the DNA sequence of cloned viral genomes, it was concluded that the protease is encoded in the viral genome itself (36,(51)(52)(53).…”