Molecular and Enzymatic Characterization of the Porcine Endogenous Retrovirus Protease

Abstract

The protease of the porcine endogenous retrovirus (PERV) subtypes A/B and C was recombinantly expressed in Escherichia coli as proteolytically active enzyme and characterized. The PERV Gag precursor was also recombinantly produced and used as the substrate in an in vitro enzyme assay in parallel with synthetic nonapeptide substrates designed according to cleavage site sequences identified in the PERV Gag precursor. The proteases of all PERV subtypes consist of 127 amino acid residues with an M r of 14,000 as revealed by determining the protease N and C termini. The PERV proteases have a high specificity for PERV substrates and do not cleave human immunodeficiency virus (HIV)-specific substrates, nor are they inhibited by specific HIV protease inhibitors. Among the known retroviral proteases, the PERV proteases resemble most closely the protease of the murine leukemia retrovirus.Organ xenotransplantation from pigs may be one possibility to solve the shortage of human organ donors. An unanswered question is, however, the risk of viral infection by porcine endogenous retroviruses (PERVs) upon xenografting (3,5,7,10,11,16). One way to minimize this problem might be the option of designing an effective antiviral chemotherapy against PERV, comparable to that presently employed against human immunodeficiency virus (HIV) infection. Here we have characterized the PERV protease as a target for protease inhibitors (PIs) that might be employed as antiviral agents for chemotherapy.Three main subtypes of PERV, A, B, and C, are known to date (14). All are classical C-type retroviruses (e.g., 1, 2, 15) and are genetically similar but not identical. We have revealed the coding sequences for the proteases of all three PERV subtypes, produced active proteases by recombinant methods in Escherichia coli, and established an in vitro assay system for PERV protease activity.Coding sequence of the PERV protease. For recombinant expression of the PERV protease, the boundaries of its coding sequence had to be revealed. Possible N and C termini of the functional PERV protease coding sequence were predicted by multiple alignment of the translated sequences of gag and pol regions of PERV subtypes A/B (DuxDL3791 [J. Blusch et al., unpublished data]) and C (PERV-MSL [1]) with the protease sequence of Moloney murine leukemia virus (MMLV [8,20]), the phylogenetically closest C-type retrovirus with a known protease protein sequence (Fig. 1).The alignment indicated that the C terminus of the PERV protease seemed to be highly similar to that of the murine leukemia virus protease; the N terminus of PERV protease, however, showed significant differences (Fig. 1). The N terminus of MMLV protease is known to overlap into the C-terminal Gag open reading frame (ORF) by 4 amino acid residues (T L D D) (21). Thus, the gag stop (amber) codon TAG (X in Fig. 1) is part of the protease coding sequences and can be read through by a Gln suppressor tRNA. An alignment of the protease N terminus emerged only in the way shown in Fig. 1; the L*A was provided as the r...

Porcine Endogenous Retrovirus (PERV) – Molecular Structure and Replication Strategy in the Context of Retroviral Infection Risk of Human Cells

“…The pol gene encodes the following enzymes: protease (PR), RT, and integrase (IN). PR is a protein with a molecular weight of 14 kDa (p14) that catalyzes the proteolysis of the Gag and Pol polyproteins into the proteins described above ( Czauderna et al, 2000 ; Blusch et al, 2002 ). RT is responsible for the transcription of viral ssRNA into dsDNA, which is subsequently incorporated into the genome of the host with the help of IN ( Denner and Tonjes, 2012 ).…”

“…In contrast to the Pol polyprotein, the products of the gag gene can be synthesized alone or as a single large polyprotein together with the products of the pol gene. The gag stop codon UAG is a part of the PR-coding sequence and can be read through by suppressor tRNA-accepting glutamine ( Akiyoshi et al, 1998 ; Blusch et al, 2002 ).…”

Promoter activity analysis and methylation characterization of LTR elements of PERVs in NIH miniature pig

“…Inhibitors of HIV reverse transcriptase could be used against the PERV reverse transcriptase (Powell et al, 2000;Qari et al, 2001). However, protease inhibitors of HIV do not exert inhibitory effects on PERV protease (Blusch et al, 2002). Thus, novel protease inhibitors might offer the most promising option for antiviral protection against the potential risks of human PERV infection.…”

New Insights into Porcine Endogenous Retrovirus (PERV)

“…In PERVs, the pol gene encodes the protease (PR), the integrase (IN), and the reverse transcriptase (RT). The PR can be auto-activated to cleave Gag and Pol at specific sites to initiate PERV maturation (Blusch, Seelmeir, & von der Helm, 2002). The RT is a key enzyme in viral propagation, generating complementary DNAs (cDNAs) from the PERV ssRNA templates.…”