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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ABSTRACT |
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MAIN TEXT |
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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, MasonPfizer 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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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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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.
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ACKNOWLEDGEMENTS |
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Received 28 January 2003;
accepted 1 May 2003.