- Email: [email protected]
Moloney Murine Leukemia Virus Retropepsin
226
Clan AA (A2) | 53. Moloney Murine Leukemia Virus Retropepsin
Luis Mene´ndez-Arias Centro de Biologı´a Molecular ‘Severo Ochoa’, (Consejo Superior de Investigaciones Cientı´ficas UAM), c/Nicola´s Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain. Email: [email protected]
Jo´zsef To¨zse´r Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Egyetem te´r 1, H-4012 Debrecen, Hungary. Email: [email protected]
Stephen Oroszlan HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702-1201, USA. Email: [email protected], [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00052-1
Chapter 53
Moloney Murine Leukemia Virus Retropepsin DATABANKS MEROPS name: murine leukemia virus-type retropepsin MEROPS classification: clan AA, family A2, subfamily A2A, peptidase A02.008 Species distribution: superkingdoms Eukaryota, Viridae Reference sequence from: Moloney murine leukemia virus (UniProt: P03355)
Name and History Early evidence of the proteolytic processing of murine leukemia virus (MLV) Gag polyproteins came from immunoprecipitation studies with Rauscher and Moloney MLV [13]. High molecular mass precursor polyproteins were immunoprecipitated with antisera made against proteins of the mature virus. The presence of a proteolytic factor in detergent-treated extracts of MLV was then reported by Yoshinaka & Luftig [4,5], who described the morphological conversion of ‘immature’ viral cores to a ‘mature’ form after addition of a partially purified P65-70 proteolytic factor [5,6]. This protease was later isolated from Moloney MLV in large quantities and its proteolytic activity was shown using a 65 kDa Gag precursor polyprotein as substrate [7]. The N- and C-terminal amino
acid sequences of the retropepsin were then determined and aligned with the primary structure deduced from the DNA sequence of Moloney MLV. It was shown that the Moloney murine leukemia virus retropepsin was encoded by the gag-pol genes and synthesized through suppression of the UAG termination codon found at the end of gag [7] and not by splicing of the viral genome, as previously assumed [8]. This enzyme is also known as MLV retropepsin or MLV protease (or proteinase).
Activity and Specificity Viral precursor polyproteins (e.g. the uncleaved 65 kDa precursor from Gazdar murine sarcoma virus) were used as substrates in assays performed with retropepsin isolated from virions [7,9]. Cleavage sites in Gag and Gag-Pol have been identified [10,11]. Synthetic peptides mimicking the maturation sites found in those polyproteins were shown to be useful substrates for Moloney MLV retropepsin [12]. Their Km values ranged from 16.6 to 98.0 µM, while the corresponding kcat values ranged from 0.07 to 0.71 s21 [12]. Proteolytic assays were done in 0.25 M potassium phosphate buffer, pH 6.0, containing 3.0 M NaCl and 0.15 mM substrate, and after incubation at 37 C products were analyzed by HPLC. Under these
Clan AA (A2) | 53. Moloney Murine Leukemia Virus Retropepsin
conditions, its optimum pH is 6.0 and its maximum activity is observed at 3.0 M NaCl concentration [12]. The optimum pH of MLV retropepsin was 5.0 in assays carried out in 50 mM 2-(N-morpholino)ethanesulfonic acid, 100 mM Tris, 50 mM sodium acetate and 1 M NaCl, and using the chromogenic substrate Lys-Ala-Arg-Val-Nle-pnitroPhe-Glu-Ala-Nle-amide (L6525; Sigma) [13]. A Gag-PR (protease) fusion protein was expressed in E. coli and shown to carry out efficient cleavage at the proper junctions. This indicates that specific host factors are not required for the virus protease to cleave its substrates [14]. Moloney MLV retropepsin is able to cleave efficiently synthetic peptides representing maturation sites found in polyproteins of other retroviruses [12,13]. For example, HIV-1 retropepsin (Chapter 44) substrate Val-Ser-GlnAsn-TyrkPro-Ile-Val-Gln is cleaved by the Moloney MLV retropepsin, with kcat and Km values of 0.55 s21 and 261 µM, respectively. Moloney MLV and HIV retropepsins showed similar substrate specificity as revealed by kinetic analysis using Val-Ser-Gln-Asn-TyrkPro-Ile-ValGln analogs with single amino acid substitutions in the P4-P30 positions [1517]. Specificity differences between MLV and HIV proteases were mainly observed at positions P4 and P2. While MLV prefers branched-chain amino acid residues (Val, Ile, Leu), HIV-1 and HIV-2 proteases preferred rather smaller, more hydrophilic residues at these positions. In contrast to the HIV-1 protease, the MLV retropepsin does not favor cleavage at the MA/ CA or Ca/p2 junctions, when Glu or Gln are present at the P20 position [18]. Site-directed mutagenesis studies suggest that His37, Val39 and Ala57 at the substratebinding pocket of Moloney MLV retropepsin are major determinants of the differences in substrate specificity between MLV and HIV retropepsins [19]. Shorter analogs of Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln that are hydrolyzed by Moloney MLV retropepsin [15] are available from Bachem. Several HIV-1 retropepsin inhibitors have been reported to be effective on MLV retropepsins, with 50% inhibitory concentrations of less than 1 µM as demonstrated using purified protease [12] as well as in cell culture assays [20,21]. In enzymatic assays carried out with a fluorescent analog of the MLV p12/CA cleavage site, amprenavir emerged as a potent inhibitor of MLV retropepsin with a Ki of 20 nM (in the same conditions, amprenavir inhibited the HIV-1 retropepsin with a Ki , 1 nM) [13].
Structural Chemistry Moloney MLV retropepsin is a homodimeric enzyme [22] formed by two polypeptides of 125 amino acids each, with calculated Mr 5 13 315 [7]. The crystal structure of
227
the related xenotropic murine leukemia virus-related virus ˚ resolution (XMRV) retropepsin has been solved at 1.97 A (PDB file 3NR6) [23]. XMRV retropepsin is closely related to Moloney MLV protease, differing only at positions 3 and 115. N- and C-terminal structural elements in the XMRV protease are longer than in most other retropepsins and provide a dimerization interface that is substantially different from those seen in other retroviral protease structures [23]. Moreover, the dimerization mode of XMRV retropepsin was found to be similar to that reported for the putative protease domain of Saccharomyces cervevisiae Ddi1 [24]. The inter-subunit ionic interactions found in HIV-1 retropepsin are substituted by hydrophobic contacts in the XMRV enzyme [23].
Preparation The Moloney MLV retropepsin has been purified from viral particles [7], although yields were very low less than 0.3 µg of protease per mg of virus. Moloney MLV retropepsin has been expressed in Escherichia coli fused to TrpE [25], glutathione S-transferase of Schistosoma japonicum [12] or maltose-binding protein [23,26]. The retropepsin has been purified to homogeneity from bacterial extracts expressing chimeric enzymes formed by glutathione S-transferase or maltose-binding protein and the MLV retropepsin. The purification yield was around 0.2 mg of protease per liter of culture [12,26]. Higher yields (0.67 mg/liter) have been obtained by using constructs containing the maltose-binding protein, the MLV retropepsin and a hexahistidine tag at the C-terminal end of the enzyme [26]. Factor Xa cleavage sites flanking the sequence of MLV protease were also introduced in these constructs. Usage of the broad-based protease inhibitor TL-3 (C2-symmetric diol inhibitor 12) [27] during the purification of XMRV retropepsin prevented autolysis almost completely, and allowed the recovery of the enzyme at high concentrations (around 8 mg/ml) [23].
Biological Aspects The Moloney MLV retropepsin coding region is located at the 50 end of the pol gene, and its first four amino acids overlap with the 30 end of the gag gene. The fifth amino acid residue is glutamine, which is inserted by suppression of the amber termination codon at the gag-pol junction [7]. The efficiency of the translational read-through is about 410% [3]. The precursor polyproteins Gag-Pol (180 kDa) and Gag (65 kDa) are cleaved by the Moloney MLV retropepsin to render the mature viral proteins of the virus. Six cleavage sites have been identified in these two polyproteins (see review by Oroszlan & Luftig [28]).
228
Clan AA (A2) | 53. Moloney Murine Leukemia Virus Retropepsin
Cleavage between p12 and CA is essential for formation of the normal core structure found in mature MLV particles [29]. During the final stage of Env protein maturation, the Moloney MLV retropepsin cleaves the transmembrane intermediate protein Pr15(E) (or TM) to p15(E) and p2(E) [12,3032]. This cleavage activates the membrane fusion capability of the MLV Env protein and appears to be essential for infectivity [33]. Removal of the 16-amino-acid long p2(E) (also known as the R peptide) enhances the fusogenic activity of Env by affecting the conformation of the ectodomain of the protein and the strength of interactions between MLV surface (SU) and TM glycoproteins [34,35]. The mechanism leading to the activation of Moloney MLV retropepsin is not known, although dimerization of Gag-Pol precursors is likely to play an important role in the process. MLV retropepsin cleaves the eukaryotic translation initiation factors 4GI and 4GII, suggesting a role of the enzyme in mediating host translational shut-off during viral infection [36]. In addition, the MLV protease cleaves murine APOBEC3 after maturation of virions [37]. This proteolytic activity could counteract the antiviral effects of APOBEC3. APOBEC3 proteins are host factors that can be packaged into viral particles and catalyze the deamination of cytosine to uracyl in retroviral cDNA.
Distinguishing Features Moloney MLV and HIV retropepsins have similar enzymological properties. However, several specific inhibitors of HIV-1 retropepsin are less effective with the MLV proteases [12,13,20,37], and differences in the cleavage rates of various synthetic peptides were reported [13,1517]. A polyclonal antiserum against the C-terminal sequence of the Moloney MLV retropepsin has been described [12].
Related Peptidases MLV retropepsins are highly conserved and most of them share more than 95% protein sequence identity with the Moloney MLV protease. Active feline LV retropepsin has been purified from virus [9]. It shares 80% identical amino acids with Moloney MLV retropepsin. All of these enzymes appear to be very similar in terms of substrate specificity as suggested by comparison of the corresponding Gag cleavage site sequences in the different viruses. The retropepsin of porcine endogenous retrovirus (PERV) subtypes A/B and C has been obtained after expression in E. coli. It is formed by two identical polypeptide chains, each of 127 amino acids, and shares about 60% sequence identity with MLV PR [38]. The purified retropepsin
cleaved the PERV Gag precursor and synthetic oligopeptides mimicking its MA-CA and CA-NC cleavage sites. However, HIV-1-specific substrates were not hydrolyzable by the PERV protease. As reported for the MLV retropepsin, the PERV protease was weakly inhibited by pepstatin A and was resistant to clinically used HIV-1 protease inhibitors, such as saquinavir, ritonavir, indinavir and nelfinavir [38].
Further Reading See Oroszlan & Luftig [26] for a detailed account of the historical events leading to the discovery of retropepsins, with one section dedicated to MLV retropepsin, and To¨zse´r [39] for a a succinct review summarizing current knowledge on substrate specificity of retroviral retropepsins.
References [1] Arcement, L.J., Karshin, W.L., Naso, R.B., Jamjoom, G., Arlinghaus, R.B. (1976). Biosynthesis of Rauscher leukemia viral proteins: presence of p30 and envelope p15 sequences in precursor polyproteins. Virology 69, 763774. [2] Barbacid, M., Stephenson, J.R., Aaronson, S.A. (1976). gag Gene of mammalian type-C RNA tumour viruses. Nature 262, 554559. [3] Jamjoom, G.A., Naso, R.B., Arlinghaus, R.B. (1977). Further characterization of intracellular precursor polyproteins of Rauscher leukemia virus. Virology 78, 1134. [4] Yoshinaka, Y., Luftig, R.B. (1977). Murine leukemia virus morphogenesis: cleavage of P70 in vitro can be accompanied by a shift from a concentrically coiled internal strand (‘immature’) to a collapsed (‘mature’) form of the virus core. Proc. Natl. Acad. Sci. USA 74, 34463450. [5] Yoshinaka, Y., Luftig, R.B. (1977). Properties of a P70 proteolytic factor of murine leukemia viruses. Cell 12, 709720. [6] Yoshinaka, Y., Luftig, R.B. (1978). Morphological conversion of ‘immature’ Rauscher leukaemia virus cores to a ‘mature’ form after addition of the P6570 (gag gene product) proteolytic factor. J. Gen. Virol. 40, 151160. [7] Yoshinaka, Y., Katoh, I., Copeland, T.D., Oroszlan, S. (1985). Murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon. Proc. Natl. Acad. Sci. USA 82, 16181622. [8] Varmus, H.E. (1985). Reverse transcriptase rides again. Nature 314, 583584. [9] Yoshinaka, Y., Katoh, I., Copeland, T.D., Oroszlan, S. (1985). Translational read-through of an amber termination codon during synthesis of feline leukemia virus protease. J. Virol. 55, 870873. [10] Oroszlan, S., Henderson, L.E., Stephenson, J.R., Copeland, T.D., Long, C.W., Ihle, J.N., Gilden, R.V. (1978). Amino- and carboxylterminal amino acid sequences of proteins coded by gag gene of murine leukemia virus. Proc. Natl. Acad. Sci. USA 75, 14041408. [11] Henderson, L.E., Sowder, R., Copeland, T.D., Smythers, G., Oroszlan, S. (1984). Quantitative separation of murine leukemia
Clan AA (A2) | 53. Moloney Murine Leukemia Virus Retropepsin
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virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products. J. Virol. 52, 492500. Mene´ndez-Arias, L., Gotte, D., Oroszlan, S. (1993). Moloney murine leukemia virus protease: bacterial expression and characterization of the purified enzyme. Virology 196, 557563. Fe´her, A., Boross, P., Sperka, T., Miklo´ssy, G., Ka´das, J., Bagossi, P., Oroszlan, S., Weber, I.T., To¨zse´r, J. (2006). Characterization of the murine leukemia virus protease and its comparison with the human immunodeficiency virus type 1 protease. J. Gen. Virol. 87, 13211330. Cannon, K., Qin, L., Schumann, G., Boeke, J.D. (1998). Moloney murine leukemia virus protease expressed in bacteria is enzymatically active. Arch. Virol. 143, 381388. Mene´ndez-Arias, L., Weber, I.T., Soss, J., Harrison, R.W., Gotte, D., Oroszlan, S. (1994). Kinetic and modeling studies of subsites S4S30 of Moloney murine leukemia virus protease. J. Biol. Chem. 269, 1679516801. Bagossi, P., Sperka, T., Fehe´r, A., Ka´das, J., Zahuczky, G., Miklo´ssy, G., Boross, P., To¨zse´r, J. (2005). Amino acid preferences for a critical substrate binding subsite of retroviral proteases in type 1 cleavage sites. J. Virol. 79, 42134218. Eizert, H., Bander, P., Bagossi, P., Sperka, T., Miklo´ssy, G., Boross, P., Weber, I.T., To¨zse´r, J. (2008). Amino acid preferences of retroviral proteases for amino-terminal positions in a type 1 cleavage site. J. Virol. 82, 1011110117. Boross, P., Bagossi, P., Copeland, T.D., Oroszlan, S., Louis, J.M., To¨zse´r, J. (1999). Effect of substrate residues on the P20 preference of retroviral proteinases. Eur. J. Biochem. 264, 921929. Mene´ndez-Arias, L., Weber, I.T., Oroszlan, S. (1995). Mutational analysis of the substrate binding pocket of murine leukemia virus protease and comparison with human immunodeficiency virus proteases. J. Biol. Chem. 270, 2916229168. Black, P.L., Downs, M.B., Lewis, M.G., Ussery, M.A., Dreyer, G.B., Petteway, S.R. Jr., Lambert, D.M. (1993). Antiretroviral activities of protease inhibitors against murine leukemia virus and simian immunodeficiency virus in tissue culture. Antimicrob. Agents Chemother. 37, 7177. Lai, M.-H.T., Tang, J., Wroblewski, V., Dee, A.G., Margolin, N., Vlahos, C., Bowdon, B., Buckheit, R., Colacino, J., Hui, K.Y. (1993). Impeded progression of Friend disease in mice by an inhibitor of retroviral proteases. J. Acquir. Immune Defic. Syndr. 6, 2431. Yoshinaka, Y., Luftig, R.B. (1980). Physicochemical characterization and specificity of the murine leukaemia virus Pr65gag proteolytic factor. J. Gen. Virol. 48, 329340. Li, M., DiMaio, F, Zhou, D., Gustchina, A., Lubkowski, J., Dauter, Z., Baker, D., Wlodawer, A. (2011). Crystal structure of XMRV protease differs from the structures of other retropepsins. Nat. Struct. Mol. Biol. 18, 227229. Sirkis, R., Gerst, J.E., Fass, D. (2006). Ddi1, a eukaryotic protein with the retroviral protease fold. J. Mol. Biol. 364, 376387. Calkins, P., Luftig, R.B. (1993). Bacterial expression and activity of the Moloney murine leukemia virus proteinase, in: Viral Proteinases as Targets for Chemotherapy, Kra¨usslich, H.-G.,
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Oroszlan, S., Wimmer, E., eds., Cold Spring Harbor, NY: Cold Spring Harbor: Laboratory Press, pp. 113116. Fe´her, A., Boross, P., Sperka, T., Oroszlan, S., To¨zse´r, J. (2004). Expression of the murine leukemia virus protease in fusion with maltose-binding protein in Escherichia coli. Protein Express. Purif. 35, 6268. Lee, T., Laco, G.S., Torbett, B.E., Fox, H.S., Lerner, D.L., Elder, J.H., Wong, C.-H. (1998). Analysis of the S3 and S3 subsite specificities of feline immunodeficiency virus (FIV) protease: development of a broad-based protease inhibitor efficacious against FIV, SIV, and HIV in vitro and ex vivo. Proc. Natl. Acad. Sci. USA 95, 939944. Oroszlan, S., Luftig, R.B. (1990). Retroviral proteinases. Curr. Top. Microbiol. Immunol. 157, 153185. Oshima, M., Muriaux, D., Mirro, J., Nagashima, K., Dryden, K., Yeager, M., Rein, A. (2004). Effects of blocking individual maturation cleavages in murine leukemia virus Gag. J. Virol. 78, 14111420. Katoh, I., Yoshinaka, Y., Rein, A., Shibuya, M., Odaka, T., Oroszlan, S. (1985). Murine leukemia virus maturation: protease region required for conversion from ‘immature’ to ‘mature’ core form and for virus infectivity. Virology 145, 280292. Crawford, S., Goff, S.P. (1985). A deletion mutation in the 50 part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins. J. Virol. 53, 899907. Schultz, A., Rein, A. (1985). Maturation of murine leukemia virus env proteins in the absence of other viral proteins. Virology 145, 335339. Rein, A., Mirro, J., Haynes, J.G., Ernst, S.M., Nagashima, K. (1994). Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein. J. Virol. 68, 17731781. Aguilar, H.C., Anderson, W.F., Cannon, P.M. (2003). Cytoplasmic tail of Moloney murine leukemia virus envelope protein influences the conformation of the extracellular domain: Implications for mechanism of action of the R peptide. J. Virol. 77, 12811291. Li, M., Li, Z.-N., Yao, Q., Yang, C., Steinhauer, D.A., Compans, R.W. (2006). Murine leukemia virus R peptide inhibits influenza virus hemagglutinin-induced membrane fusion. J. Virol. 80, 61066114. ´ lvarez, E., Mene´ndez-Arias, L., Carrasco, L. (2003). The eukaryA otic translation initiation factor 4GI is cleaved by different retroviral proteases. J. Virol. 77, 1239212400. Abudu, A., Takaori-Kondo, A., Izumi, T., Shirakawa, K., Kobayashi, M., Sasada, A., Fukunaga, K., Uchiyama, T. (2006). Murine retrovirus escapes from murine APOBEC3 via two distinct novel mechanisms. Curr. Biol. 16, 15651570. Blusch, J.H., Seelmeir, S., von der Helm, K. (2002). Molecular and enzymatic characterization of the porcine endogenous retrovirus protease. J. Virol. 76, 79137917. To¨zse´r, J. (2010). Comparative studies on retroviral proteases: Substrate specificity. Viruses 2, 147165.
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Clan AA (A2) | 53. Moloney Murine Leukemia Virus Retropepsin
Luis Mene´ndez-Arias Centro de Biologı´a Molecular ‘Severo Ochoa’, (Consejo Superior de Investigaciones Cientı´ficas UAM), c/ Nicola´s Cabrera, 1 Campus de Cantoblanco 28049, Madrid, Spain. Email: [email protected]
Jo´zsef To¨zse´r Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Egyetem te´r 1, H-4012 Debrecen, Hungary. Email: [email protected]
Stephen Oroszlan HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702-1201, USA. Email: [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00053-3
Clan AA (A2) | 53. Moloney Murine Leukemia Virus Retropepsin
Luis Mene´ndez-Arias Centro de Biologı´a Molecular ‘Severo Ochoa’, (Consejo Superior de Investigaciones Cientı´ficas UAM), c/Nicola´s Cabrera, 1, Campus de Cantoblanco, 28049 Madrid, Spain. Email: [email protected]
Jo´zsef To¨zse´r Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Egyetem te´r 1, H-4012 Debrecen, Hungary. Email: [email protected]
Stephen Oroszlan HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702-1201, USA. Email: [email protected], [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00052-1
Chapter 53
Moloney Murine Leukemia Virus Retropepsin DATABANKS MEROPS name: murine leukemia virus-type retropepsin MEROPS classification: clan AA, family A2, subfamily A2A, peptidase A02.008 Species distribution: superkingdoms Eukaryota, Viridae Reference sequence from: Moloney murine leukemia virus (UniProt: P03355)
Name and History Early evidence of the proteolytic processing of murine leukemia virus (MLV) Gag polyproteins came from immunoprecipitation studies with Rauscher and Moloney MLV [13]. High molecular mass precursor polyproteins were immunoprecipitated with antisera made against proteins of the mature virus. The presence of a proteolytic factor in detergent-treated extracts of MLV was then reported by Yoshinaka & Luftig [4,5], who described the morphological conversion of ‘immature’ viral cores to a ‘mature’ form after addition of a partially purified P65-70 proteolytic factor [5,6]. This protease was later isolated from Moloney MLV in large quantities and its proteolytic activity was shown using a 65 kDa Gag precursor polyprotein as substrate [7]. The N- and C-terminal amino
acid sequences of the retropepsin were then determined and aligned with the primary structure deduced from the DNA sequence of Moloney MLV. It was shown that the Moloney murine leukemia virus retropepsin was encoded by the gag-pol genes and synthesized through suppression of the UAG termination codon found at the end of gag [7] and not by splicing of the viral genome, as previously assumed [8]. This enzyme is also known as MLV retropepsin or MLV protease (or proteinase).
Activity and Specificity Viral precursor polyproteins (e.g. the uncleaved 65 kDa precursor from Gazdar murine sarcoma virus) were used as substrates in assays performed with retropepsin isolated from virions [7,9]. Cleavage sites in Gag and Gag-Pol have been identified [10,11]. Synthetic peptides mimicking the maturation sites found in those polyproteins were shown to be useful substrates for Moloney MLV retropepsin [12]. Their Km values ranged from 16.6 to 98.0 µM, while the corresponding kcat values ranged from 0.07 to 0.71 s21 [12]. Proteolytic assays were done in 0.25 M potassium phosphate buffer, pH 6.0, containing 3.0 M NaCl and 0.15 mM substrate, and after incubation at 37 C products were analyzed by HPLC. Under these
Clan AA (A2) | 53. Moloney Murine Leukemia Virus Retropepsin
conditions, its optimum pH is 6.0 and its maximum activity is observed at 3.0 M NaCl concentration [12]. The optimum pH of MLV retropepsin was 5.0 in assays carried out in 50 mM 2-(N-morpholino)ethanesulfonic acid, 100 mM Tris, 50 mM sodium acetate and 1 M NaCl, and using the chromogenic substrate Lys-Ala-Arg-Val-Nle-pnitroPhe-Glu-Ala-Nle-amide (L6525; Sigma) [13]. A Gag-PR (protease) fusion protein was expressed in E. coli and shown to carry out efficient cleavage at the proper junctions. This indicates that specific host factors are not required for the virus protease to cleave its substrates [14]. Moloney MLV retropepsin is able to cleave efficiently synthetic peptides representing maturation sites found in polyproteins of other retroviruses [12,13]. For example, HIV-1 retropepsin (Chapter 44) substrate Val-Ser-GlnAsn-TyrkPro-Ile-Val-Gln is cleaved by the Moloney MLV retropepsin, with kcat and Km values of 0.55 s21 and 261 µM, respectively. Moloney MLV and HIV retropepsins showed similar substrate specificity as revealed by kinetic analysis using Val-Ser-Gln-Asn-TyrkPro-Ile-ValGln analogs with single amino acid substitutions in the P4-P30 positions [1517]. Specificity differences between MLV and HIV proteases were mainly observed at positions P4 and P2. While MLV prefers branched-chain amino acid residues (Val, Ile, Leu), HIV-1 and HIV-2 proteases preferred rather smaller, more hydrophilic residues at these positions. In contrast to the HIV-1 protease, the MLV retropepsin does not favor cleavage at the MA/ CA or Ca/p2 junctions, when Glu or Gln are present at the P20 position [18]. Site-directed mutagenesis studies suggest that His37, Val39 and Ala57 at the substratebinding pocket of Moloney MLV retropepsin are major determinants of the differences in substrate specificity between MLV and HIV retropepsins [19]. Shorter analogs of Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln that are hydrolyzed by Moloney MLV retropepsin [15] are available from Bachem. Several HIV-1 retropepsin inhibitors have been reported to be effective on MLV retropepsins, with 50% inhibitory concentrations of less than 1 µM as demonstrated using purified protease [12] as well as in cell culture assays [20,21]. In enzymatic assays carried out with a fluorescent analog of the MLV p12/CA cleavage site, amprenavir emerged as a potent inhibitor of MLV retropepsin with a Ki of 20 nM (in the same conditions, amprenavir inhibited the HIV-1 retropepsin with a Ki , 1 nM) [13].
Structural Chemistry Moloney MLV retropepsin is a homodimeric enzyme [22] formed by two polypeptides of 125 amino acids each, with calculated Mr 5 13 315 [7]. The crystal structure of
227
the related xenotropic murine leukemia virus-related virus ˚ resolution (XMRV) retropepsin has been solved at 1.97 A (PDB file 3NR6) [23]. XMRV retropepsin is closely related to Moloney MLV protease, differing only at positions 3 and 115. N- and C-terminal structural elements in the XMRV protease are longer than in most other retropepsins and provide a dimerization interface that is substantially different from those seen in other retroviral protease structures [23]. Moreover, the dimerization mode of XMRV retropepsin was found to be similar to that reported for the putative protease domain of Saccharomyces cervevisiae Ddi1 [24]. The inter-subunit ionic interactions found in HIV-1 retropepsin are substituted by hydrophobic contacts in the XMRV enzyme [23].
Preparation The Moloney MLV retropepsin has been purified from viral particles [7], although yields were very low less than 0.3 µg of protease per mg of virus. Moloney MLV retropepsin has been expressed in Escherichia coli fused to TrpE [25], glutathione S-transferase of Schistosoma japonicum [12] or maltose-binding protein [23,26]. The retropepsin has been purified to homogeneity from bacterial extracts expressing chimeric enzymes formed by glutathione S-transferase or maltose-binding protein and the MLV retropepsin. The purification yield was around 0.2 mg of protease per liter of culture [12,26]. Higher yields (0.67 mg/liter) have been obtained by using constructs containing the maltose-binding protein, the MLV retropepsin and a hexahistidine tag at the C-terminal end of the enzyme [26]. Factor Xa cleavage sites flanking the sequence of MLV protease were also introduced in these constructs. Usage of the broad-based protease inhibitor TL-3 (C2-symmetric diol inhibitor 12) [27] during the purification of XMRV retropepsin prevented autolysis almost completely, and allowed the recovery of the enzyme at high concentrations (around 8 mg/ml) [23].
Biological Aspects The Moloney MLV retropepsin coding region is located at the 50 end of the pol gene, and its first four amino acids overlap with the 30 end of the gag gene. The fifth amino acid residue is glutamine, which is inserted by suppression of the amber termination codon at the gag-pol junction [7]. The efficiency of the translational read-through is about 410% [3]. The precursor polyproteins Gag-Pol (180 kDa) and Gag (65 kDa) are cleaved by the Moloney MLV retropepsin to render the mature viral proteins of the virus. Six cleavage sites have been identified in these two polyproteins (see review by Oroszlan & Luftig [28]).
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Clan AA (A2) | 53. Moloney Murine Leukemia Virus Retropepsin
Cleavage between p12 and CA is essential for formation of the normal core structure found in mature MLV particles [29]. During the final stage of Env protein maturation, the Moloney MLV retropepsin cleaves the transmembrane intermediate protein Pr15(E) (or TM) to p15(E) and p2(E) [12,3032]. This cleavage activates the membrane fusion capability of the MLV Env protein and appears to be essential for infectivity [33]. Removal of the 16-amino-acid long p2(E) (also known as the R peptide) enhances the fusogenic activity of Env by affecting the conformation of the ectodomain of the protein and the strength of interactions between MLV surface (SU) and TM glycoproteins [34,35]. The mechanism leading to the activation of Moloney MLV retropepsin is not known, although dimerization of Gag-Pol precursors is likely to play an important role in the process. MLV retropepsin cleaves the eukaryotic translation initiation factors 4GI and 4GII, suggesting a role of the enzyme in mediating host translational shut-off during viral infection [36]. In addition, the MLV protease cleaves murine APOBEC3 after maturation of virions [37]. This proteolytic activity could counteract the antiviral effects of APOBEC3. APOBEC3 proteins are host factors that can be packaged into viral particles and catalyze the deamination of cytosine to uracyl in retroviral cDNA.
Distinguishing Features Moloney MLV and HIV retropepsins have similar enzymological properties. However, several specific inhibitors of HIV-1 retropepsin are less effective with the MLV proteases [12,13,20,37], and differences in the cleavage rates of various synthetic peptides were reported [13,1517]. A polyclonal antiserum against the C-terminal sequence of the Moloney MLV retropepsin has been described [12].
Related Peptidases MLV retropepsins are highly conserved and most of them share more than 95% protein sequence identity with the Moloney MLV protease. Active feline LV retropepsin has been purified from virus [9]. It shares 80% identical amino acids with Moloney MLV retropepsin. All of these enzymes appear to be very similar in terms of substrate specificity as suggested by comparison of the corresponding Gag cleavage site sequences in the different viruses. The retropepsin of porcine endogenous retrovirus (PERV) subtypes A/B and C has been obtained after expression in E. coli. It is formed by two identical polypeptide chains, each of 127 amino acids, and shares about 60% sequence identity with MLV PR [38]. The purified retropepsin
cleaved the PERV Gag precursor and synthetic oligopeptides mimicking its MA-CA and CA-NC cleavage sites. However, HIV-1-specific substrates were not hydrolyzable by the PERV protease. As reported for the MLV retropepsin, the PERV protease was weakly inhibited by pepstatin A and was resistant to clinically used HIV-1 protease inhibitors, such as saquinavir, ritonavir, indinavir and nelfinavir [38].
Further Reading See Oroszlan & Luftig [26] for a detailed account of the historical events leading to the discovery of retropepsins, with one section dedicated to MLV retropepsin, and To¨zse´r [39] for a a succinct review summarizing current knowledge on substrate specificity of retroviral retropepsins.
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Luis Mene´ndez-Arias Centro de Biologı´a Molecular ‘Severo Ochoa’, (Consejo Superior de Investigaciones Cientı´ficas UAM), c/ Nicola´s Cabrera, 1 Campus de Cantoblanco 28049, Madrid, Spain. Email: [email protected]
Jo´zsef To¨zse´r Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Egyetem te´r 1, H-4012 Debrecen, Hungary. Email: [email protected]
Stephen Oroszlan HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702-1201, USA. Email: [email protected] Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00053-3