Mutational analysis of the R peptide cleavage site of Moloney murine leukaemia virus envelope protein

Yoshinao Kubo1,2 and Hiroshi Amanuma1

1 Molecular Cell Science Laboratory, RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan
2 Department of Preventive Medicine and AIDS Research, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan

Correspondence
Yoshinao Kubo (at Institute of Tropical Medicine)
yoshinao{at}net.nagasaki-u.ac.jp


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Moloney murine leukaemia virus (MoMLV) enters host cells by membrane fusion between the viral envelope and the host cell membrane. The cytoplasmic tail (R peptide) of the MoMLV envelope protein (Env) is cleaved by the viral protease during virion maturation. R peptide-truncated Env induces syncytia in susceptible cells but R peptide-containing Env does not, indicating that the R peptide inhibits membrane fusion. To examine the function of amino acid residues at the R peptide cleavage site in virus entry, mutant Env expression plasmids containing amino acid substitutions at these cleavage site residues were constructed. Some of these mutants induced syncytia in NIH 3T3 cells, even though they expressed the R peptide, indicating the importance of these residues for membrane fusion inhibition by the R peptide. Some mutants in which R peptide cleavage was detected had comparable transduction efficiency to wild-type Env, but mutants in which R peptide cleavage was not detected had lower transduction efficiency than wild-type Env. This result strongly supports that R peptide cleavage is required for virus entry.


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Moloney murine leukaemia virus (MoMLV) entry into host cells is mediated by fusion between the viral envelope and host cell membrane after receptor recognition (Albritton et al., 1989). These reactions are catalysed by the envelope glycoprotein (Env) encoded by the viral genome. The Env protein of MoMLV is synthesized as a gp85 precursor protein and is cleaved to cell surface (SU) and transmembrane (TM) subunits during its transport to the cell surface (Schultz & Rein, 1985). The SU and TM proteins are involved in binding to the cellular receptor (Battini et al., 1992; Davey et al., 1999; MacKrell et al., 1996; Ott & Rein, 1992; Zavorotinskaya & Albritton, 1999) and subsequent fusion between the viral envelope and host cell membrane (Januszeski et al., 1997; Jones & Risser, 1993; Zhao et al., 1998), respectively.

The C-terminal 16 aa (R peptide) of the TM protein are further cleaved by the viral protease during virion maturation (Green et al., 1981; Henderson et al., 1984). R peptide-truncated Env protein (R- Env) has an enhanced ability to induce membrane fusion compared to Env protein containing the R peptide (R+ Env). This indicates that the R peptide inhibits membrane fusion (Januszeski et al., 1997; Kiernan & Freed, 1998; Li et al., 2001; Melikyan et al., 2000; Ragheb & Anderson, 1994; Rein et al., 1994; Thomas et al., 1997; Yang & Compans, 1996, 1997). The cytoplasmic tails of the Env proteins of other retroviruses (for example, Mason–Pfizer monkey virus, equine infectious anaemia virus, spleen necrosis virus, gibbon ape leukaemia virus and porcine endogenous retrovirus) are also cleaved by viral protease and their products have higher fusogenicity than the non-processed Env proteins, similar to the R peptide of MoMLV (Rice et al., 1990; Brody et al., 1994; Bobkova et al., 2002). It would be advantageous for the virus to delay membrane fusion activity until the virus leaves the infected cell, as fusogenic Env protein kills virus-producing cells through syncytium formation. Recently, Aguilar et al. (2003) have reported that the R peptide influences the conformation of the extracellular domain of the TM subunit.

To understand the role of the R peptide cleavage site of MoMLV Env protein in syncytium formation, incorporation into virus particles, R peptide cleavage by the viral protease and entry into host cells, plasmids encoding mutant Env proteins containing amino acid substitutions at the R peptide cleavage site were constructed by PCR-mediated mutagenesis (Cheng et al., 1994; Higuchi et al., 1988; Kubo et al., 1994). The leucine residue at the N-terminal side of the R peptide cleavage site (position 616) was changed to arginine (L616R), alanine (L616A), valine (L616V) and isoleucine (L616I). The valine residue at the C-terminal side of the R peptide cleavage site (position 617) was changed to translation termination (R-), arginine (V617R), alanine (V617A), leucine (V617L) and isoleucine (V617I).

To examine the fusogenicity of these mutant Env proteins, 293T cells were transfected with these mutant Env expression plasmids using Trans IT LT1 Polyamine reagent (Mirus). NIH 3T3 cells were then added. The R-, L616R, L616A, V617R, V617A and V617L mutants induced syncytia (Table 1). However, syncytia were not detected in wild-type-, L616V-, L616I- and V617I-transfected cells. This result indicates that the residues at the R peptide cleavage site are important for the inhibition of membrane fusion by the R peptide.


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Table 1. Characteristics of MoMLV vectors expressing mutant Env proteins

 
To determine the transduction titres of MoMLV vectors carrying the mutant Env proteins, TELCeB6 pre-packaging cells (Cosset et al., 1995) were transfected with the mutant Env expression plasmids. The expression of mutant Env proteins in transfected TELCeB6 cells was analysed by Western immunoblotting using anti-SU antiserum. Similar amounts of Env proteins were detected in the transfected TELCeB6 cells, indicating that the amino acid substitutions have no effect on Env protein expression (data not shown). The precursor Env protein detected in TELCeB6 cells transfected with the R- Env expression plasmid was slightly smaller than that detected in cells transfected with the other expression plasmids. This result confirms the truncation of the R peptide in the R- Env expression construct. Western immunoblotting of cell lysates prepared from the transfected TELCeB6 cells was performed using an antiserum which recognizes the capsid (CA) subunit of the MoMLV Gag protein. Equal amounts of precursor Gag and mature CA proteins were detected in all transfected cells. This result indicates that the cell lysates analysed contain equal amounts of protein.

Virions were pelleted from the culture supernatant of transfected TELCeB6 cells by ultracentrifugation through 20 % sucrose. Western immunoblotting using the anti-SU antiserum was performed on the virion preparations. Reduced levels of the mature SU protein were detected in virion preparations from the R--, L616R- and V617R-transfected cells compared to those from cells transfected with the wild-type Env expression plasmid (Fig. 1A). Equal amounts of the mature CA protein, however, were detected in all virion preparations, indicating that the preparations contain equal amounts of virion. The precursor Env and Gag proteins were not detected in the virion samples, confirming the virion preparation and minimal contamination of cells. These results indicate that the R peptide truncation and the amino acid substitutions of the residues at positions 616 and 617 by arginine (L616R and V617R) impair the incorporation of Env protein into virions.



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Fig. 1. Western blot analysis of viral proteins. TELCeB6 cells were transfected with the non-tagged mutant Env expression plasmids. Virus particles were pelleted by ultracentrifugation of the culture supernatant (A). Western blotting using the anti-SU (upper panel) and the anti-CA (lower panel) antiserum was then performed. TELCeB6 cells were transfected with the HA-tagged mutant Env expression plasmids. Cell lysates prepared from the transfected cells were subjected to Tris/Tricine-PAGE and Western blotting using the anti-HA antibody (B).

 
To determine the transduction titres of MoMLV vectors expressing mutant Env proteins, culture supernatants of TELCeB6 cells transfected with these mutant Env expression plasmids were inoculated to NIH 3T3 and XC cells in the presence of polybrene (4 µg ml-1). Cells were then stained with X-Gal. The transduction titre of a retrovirus vector carrying the wild-type Env protein was 1–8x104 c.f.u. ml-1. The transduction titre of a MoMLV vector carrying the R- Env protein was about 1/10 times lower than that of the wild-type Env protein (Fig. 2A). Titres of the L616V, L616I, L616R, V617I and V617R Env proteins were 5–25 % of that of the wild-type Env protein. Titres of the L616A, V617A and V617L Env proteins were, however, relatively high and 70–80 % of that of the wild-type Env protein.



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Fig. 2. Transduction titres of MoMLV vectors expressing mutant Env protein. TELCeB6 cells were transfected with the non-tagged mutant Env expression plasmids (A). NIH 3T3 (upper panel) and XC cells (lower panel) were inoculated with culture supernatants of the transfected cells. Relative values to transduction titre by wild-type Env protein are indicated. The titre of the wild-type MoMLV vector was 1–8x104 c.f.u. ml-1. Transduction titres in NIH 3T3 cells by the HA-tagged mutant Env proteins (wild-type-HA) are indicated in (B). Relative values to transduction titre by the wild-type Env protein tagged with HA (wild-type-HA) are indicated. The titre by wild-type-HA was 9x103 to 5x104 c.f.u. ml-1.

 
Expression plasmids encoding mutant Env proteins C-terminally tagged with the influenza virus haemagglutinin (HA) epitope were constructed to detect the cleaved R peptide. These HA-tagged mutant Env expression plasmids were transfected to TELCeB6 cells and their transduction titres were measured in NIH 3T3 cells. As shown in Fig. 2(B), transduction titres by MoMLV vectors expressing the L616A-HA, V617A-HA and V617L-HA Env proteins were comparable to that of a vector expressing the wild-type Env protein C-terminally tagged with the HA epitope (wild-type-HA). Transduction titres of vectors expressing the other mutant Env proteins tagged with the HA epitope, L616R-HA, L616V-HA, L616I-HA, V617R-HA and V617I-HA, were less than 25 % of that of the vector expressing wild-type-HA. These results obtained from the HA-tagged Env proteins was consistent with those from the non-tagged Env proteins (Fig. 2A), indicating that the C-terminal tagging of the Env protein by the HA epitope had little effect on Env protein function. Furthermore, fusogenicity of these HA-tagged mutant Env proteins was similar to that of the untagged mutant Env proteins (data not shown).

To analyse the R peptide cleavage of the HA-tagged mutant Env proteins, TELCeB6 cells were transfected with the HA-tagged mutant Env expression plasmids. Cell lysates were subjected to Tris/Tricine-PAGE (15 %) and then Western immunoblotting was performed with an anti-HA epitope antibody (Covance). Because the HA-tagged R peptide has only 24 aa, Tris/Tricine-PAGE (Schagger & von Jagow, 1987) was performed. Cleaved R peptides tagged with the HA epitope were detected in cells transfected with wild-type-HA, L616A-HA, V617A-HA and V617L-HA Env expression plasmids but not with the L616R-HA, L616V-HA, L616I-HA, V617R-HA and V617I-HA Env expression plasmids (Fig. 1B). Equal amounts of the precursor Env and TM proteins tagged with the HA epitope were detected in all cell lysates analysed. This result indicates that R peptide cleavage of the L616A-HA, V617A-HA and V617L-HA Env proteins occurs as efficiently as that of the wild-type-HA, but those of L616R-HA, L616V-HA, L616I-HA, V617R-HA and V617I-HA Env proteins do not.

The L616R mutant constructed in this study induced syncytia (Table 1), although Rein et al. (1994) have reported that the same mutant does not. Four independent L616R expression plasmids constructed by independent PCR, however, all induced syncytia in our study. Nucleotide sequences of the L616R expression plasmids were determined and no unexpected mutations were detected. Rein et al. (1994) used CHO cells as donor cells expressing Env protein, while 293T cells were used in our study. When CHO, mink lung and HeLa cells were used as donor cells, the L616R Env protein also induced syncytia by mixed culture with NIH 3T3 cells (data not shown). The amino acid substitution of hydrophobic leucine by basic arginine in the L616R mutant Env should induce a dramatic change in the three-dimensional structure around the mutated site and suppress R peptide function to inhibit membrane fusion. The L616R, L616A, V617R, V617A and V617L mutant Env proteins induced syncytia in NIH 3T3 cells (Table 1). It has been reported that mutations of the leucine residue at position 618 make the Env protein fusogenic (Yang & Compans, 1997). These results suggested that the amino acid residues at positions 616, 617 and 618 are important for the inhibition of syncytium formation by the R peptide.

To detect the R peptide cleavage of the mutant Env proteins, plasmids encoding mutant Env proteins C-terminally tagged with the HA epitope were constructed. Epitope tagging did not affect transduction efficiency (Fig. 2B) and fusogenicity of the Env proteins. It has been reported that linker insertions around the C-terminal region of the R peptide of MoMLV Env protein do not affect their surface expression and transduction efficiency (Rothenberg et al., 2001). This finding is consistent with our result. Therefore, it is unlikely that the C-terminal HA tagging of the Env protein affects the R peptide cleavage. R peptide cleavage of the L616R, L616V, L616I, V617R and V617I mutant Env proteins was not detected (Fig. 1B). It has been reported using synthetic peptides as protease substrates that amino acid sequences recognized by MoMLV protease are not so specific but that hydrophobic amino acids are involved (Boross et al., 1999; Menendez-Arias et al., 1993, 1994). Therefore, it was interesting that R peptide cleavage of the L616V, L616I and V617I Env proteins was not detected, as these amino acids are also hydrophobic. Granowitz et al. (1996) have reported that R peptide cleavage of L616I is defective, as was seen in our study. The defect in the R peptide cleavage of the L616R and V617R mutant Env proteins could be due to the amino acid substitution of the hydrophobic leucine and valine residues by basic arginine or the impaired incorporation of the L616R and V617R Env proteins into virions, as R peptide cleavage occurs after incorporation of Env protein into virions (Green et al., 1981; Henderson et al., 1984).

These results are summarized in Table 1. MoMLV vectors carrying the L616A, V617A and V617L mutant Env proteins in which R peptide cleavage occurred showed comparable transduction titres to the wild-type MoMLV vector. Vectors expressing the L616V, L616I and V617I Env proteins in which the R peptide cleavage was not detected showed much lower transduction titres than the wild-type vector. This result strongly supports that R peptide cleavage is necessary for efficient transduction by the Env protein.

R peptide cleavage of the L616V, L616I and V617I Env proteins was not detected (Fig. 1B). These Env proteins induced syncytia in XC cells but not in NIH 3T3 cells, even though they have the R peptide (Jones & Risser, 1993; Kubo et al., 2002). This suggests that the vectors with these mutant Env proteins specifically transduce XC cells, but, like NIH 3T3 cells, they did not (Fig. 2A). This result suggests that the syncytium formation in XC cells by the R+ Env protein is not associated with the membrane fusion required for virus entry into cells.

The results reported here indicate that the amino acid residues at the R peptide cleavage site are important for inhibition of membrane fusion by the R peptide as well as for R peptide cleavage by the viral protease. These results also strongly support the previous finding that the R peptide cleavage of the Env protein is required for virus entry into host cells.


   ACKNOWLEDGEMENTS
 
We thank F.-L. Cosset for TELCeB6 cells, A. Rein for an anti-TM antiserum and A. Ishimoto, H. Sato and N. Yamamoto for discussion. We also thank R. Fujita, M. Katane, E. Takao and N. Sasaki for assistance and A. Koshiyama for secretarial support. This work was supported by the Gene Science Research grant of RIKEN to H. Amanuma. Y. Kubo was a special research fellow of RIKEN.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
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, 1281–1291.[CrossRef][Medline]

Albritton, L. M., Tseng, L., Scadden, D. & Cunningham, J. M. (1989). A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57, 659–666.[Medline]

Battini, J.-L., Heard, J. M. & Danos, O. (1992). Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses. J Virol 66, 1468–1475.[Abstract]

Bobkova, M., Stitz, J., Engelstadter, M., Cichutek, K. & Buchholz, C. J. (2002). Identification of R-peptides in envelope proteins of C-type retroviruses. J Gen Virol 83, 2241–2246.[Abstract/Free Full Text]

Boross, P., Bagossi, P., Copeland, T. D., Oroszlan, S., Louis, J. M. & Tozser, J. (1999). Effect of substrate residues on the P2' preference of retroviral proteinases. Eur J Biochem 264, 921–929.[Abstract/Free Full Text]

Brody, B. A., Rhee, S. G. & Hunter, E. (1994). Postassembly cleavage of a retroviral glycoprotein cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. J Virol 68, 4620–4627.[Abstract]

Cheng, S., Fockler, C., Barnes, W. M. & Higuchi, R. (1994). Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci U S A 91, 5695–5699.[Abstract]

Cosset, F.-L., Takeuchi, Y., Battini, J.-L., Weiss, R. A. & Collins, M. K. L. (1995). High-titer packaging cells producing recombinant retroviruses resistant to human serum. J Virol 69, 7430–7436.[Abstract]

Davey, R. A., Zuo, Y. & Cunningham, J. M. (1999). Identification of a receptor-binding pocket on the envelope protein of Friend murine leukemia virus. J Virol 73, 3758–3763.[Abstract/Free Full Text]

Granowitz, C., Berkowitz, R. D. & Goff, S. P. (1996). Mutations affecting the cytoplasmic domain of the Moloney murine leukemia virus envelope protein: rapid reversion during replication. Virus Res 41, 25–42.[CrossRef][Medline]

Green, N., Shinnick, T. M., Witte, O., Ponticelli, A., Sutcliffe, J. G. & Lerner, R. A. (1981). Sequence-specific antibodies show that maturation of Moloney leukemia virus envelope polyprotein involves removal of a COOH-terminal peptide. Proc Natl Acad Sci U S A 78, 6023–6027.[Abstract]

Henderson, L. E., Sowder, R., Copeland, T. D., Smythers, G. & Oroszlan, S. (1984). Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described Gag and Env cleavage products. J Virol 52, 492–500.[Medline]

Higuchi, R., Krummel, B. & Saiki, R. K. (1988). A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 16, 7351–7367.[Abstract]

Januszeski, M. M., Cannon, P. M., Chen, D., Rozenberg, Y. & Anderson, W. F. (1997). Functional analysis of the cytoplasmic tail of Moloney murine leukemia virus envelope protein. J Virol 71, 3613–3619.[Abstract]

Jones, J. S. & Risser, R. (1993). Cell fusion induced by the murine leukemia virus envelope glycoprotein. J Virol 67, 67–74.[Abstract]

Kiernan, R. E. & Freed, E. O. (1998). Cleavage of the murine leukemia virus transmembrane Env protein by human immunodeficiency virus type 1 protease: transdominant inhibition by matrix mutations. J Virol 72, 9621–9627.[Abstract/Free Full Text]

Kubo, Y., Kakimi, K., Higo, K., Wang, L., Kobayashi, H., Kuribayashi, K., Masuda, T., Hirama, T. & Ishimoto, A. (1994). The p15gag and p12gag regions are both necessary for the pathogenicity of the murine AIDS virus. J Virol 68, 5532–5537.[Abstract]

Kubo, Y., Ono, T., Ogura, M., Ishimoto, A. & Amanuma, H. (2002). A glycosylation-defective variant of the ecotropic murine retrovirus receptor is expressed in rat XC cells. Virology 303, 338–344.[CrossRef][Medline]

Li, M., Yang, C. & Compans, R. W. (2001). Mutations in the cytoplasmic tail of murine leukemia virus envelope protein suppress fusion inhibition by R peptide. J Virol 75, 2337–2344.[Abstract/Free Full Text]

MacKrell, A. J., Soong, N. W., Curtis, C. M. & Anderson, W. F. (1996). Identification of a subdomain in the Moloney murine leukemia virus envelope protein involved in receptor binding. J Virol 70, 1768–1774.[Abstract]

Melikyan, G. B., Markosyan, R. M., Brener, S. A., Rozenberg, Y. & Cohen, F. S. (2000). Role of the cytoplasmic tail of ecotropic Moloney murine leukemia virus Env protein in fusion pore formation. J Virol 74, 447–455.[Abstract/Free Full Text]

Menendez-Arias, L., Gotte, D. & Oroszlan, S. (1993). Moloney murine leukemia virus protease: bacterial expression and characterization of the purified enzyme. Virology 196, 557–563.[CrossRef][Medline]

Menendez-Arias, L., Weber, I. T., Soss, J., Harrison, R. W., Gotte, D. & Oroszlan, S. (1994). Kinetic and modeling studies of subsites S4–S3' of Moloney murine leukemia virus protease. J Biol Chem 269, 16795–16801.[Abstract/Free Full Text]

Ott, D. & Rein, A. (1992). Basis for receptor specificity of nonecotropic murine leukemia virus surface glycoprotein gp70SU. J Virol 66, 4632–4638.[Abstract]

Ragheb, J. A. & Anderson, W. F. (1994). pH-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry. J Virol 68, 3220–3231.[Abstract]

Rein, A., Mirro, J., Haynes, J. G., Ernst, S. M. & Nagashima, K. (1994). Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p12E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein. J Virol 68, 1773–1781.[Abstract]

Rice, N. R., Henderson, L. E., Sowder, R. C., Copeland, T. D., Oroszlan, S. & Edwards, J. F. (1990). Synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus. J Virol 64, 3770–3778.[Medline]

Rothenberg, S. M., Olsen, M. N., Laurent, L. C., Crowley, R. A. & Brown, P. O. (2001). Comprehensive mutational analysis of the Moloney murine leukemia virus envelope protein. J Virol 75, 11851–11862.[Abstract/Free Full Text]

Schagger, H. & von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368–379.[Medline]

Schultz, A. & Rein, A. (1985). Maturation of murine leukemia virus Env proteins in the absence of other viral proteins. Virology 145, 335–339.[CrossRef][Medline]

Thomas, A., Gray, K. D. & Roth, M. J. (1997). Analysis of mutations within the cytoplasmic domain of the Moloney murine leukemia virus transmembrane protein. Virology 227, 305–313.[CrossRef][Medline]

Yang, C. & Compans, R. W. (1996). Analysis of the cell fusion activities of chimeric simian immunodeficiency virus-murine leukemia virus envelope proteins: inhibitory effects of the R peptide. J Virol 70, 248–254.[Abstract]

Yang, C. & Compans, R. W. (1997). Analysis of the murine leukemia virus R peptide: delineation of the molecular determinants which are important for its fusion inhibition activity. J Virol 71, 8490–8496.[Abstract]

Zavorotinskaya, T. & Albritton, L. M. (1999). A hydrophobic patch in ecotropic murine leukemia virus envelope protein is the putative binding site for a critical tyrosine residue on the cellular receptor. J Virol 73, 10164–10172.[Abstract/Free Full Text]

Zhao, Y., Zhu, L., Benedict, C. A., Chen, D., Anderson, W. F. & Cannon, P. M. (1998). Functional domains in the retroviral transmembrane protein. J Virol 72, 5392–5398.[Abstract/Free Full Text]

Received 28 January 2003; accepted 1 May 2003.