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In addition to PTAP L domains, primate lentiviruses carry Alix-binding motifs that include the recently described type 3 SREKPYKEVTEDLLHLNSLF sequence. We examined the requirements for the type 3 sequence motif in simian immunodeficiency virus SIVsmE543 and identified the 499LNSLF503 sequence as a key functional determinant. Mutation of distal leucines 499L and 502L (LL mutant) caused an inhibitory effect on Alix-dependent SIVsmE543 release that was quantitatively similar to that observed following disruption of the type 3 L domain or RNA interference (RNAi) depletion of Alix. Similar results were obtained with the SIVmac239 LL mutant. Thus, distal leucines are key determinants of SIVsmE543 and SIVmac239 type 3 L domains.
Retroviruses acquire their envelopes at the host cell membrane of infected cells. To this end, they utilize short sequences designated L (late) domains to recruit members of the endosomal sorting complex required for transport (ESCRT) to catalyze membrane modeling events that lead to virus release (1, 2, 5, 11, 16). Three types of L domains have so far been identified: the PT/SAP, PPXY, and LYPXnL motifs. They bind the host proteins Tsg101, Nedd4-like ubiquitin ligase family members, and Alix, respectively (6, 13, 15, 23, 25, 26). These interactions lead to the recruitment of members of the ESCRT pathway and require the activity of the AAA ATPase VPS4 (14, 23, 24, 26).
All retroviral Gag proteins contain at least one L domain, though most carry multiple L domain motifs, believed to function synergistically and ensure efficient viral release (4, 7, 8, 13, 21, 27). Structural studies have been used to determine L domain sequence and functional requirements. Only limited sequence variability was noted in motifs that bind Tsg101, including PTAP, PSAP, and PXXP motifs found in Hrs, the natural partner for Tsg101 (3, 10, 18–20, 28). However, more sequence divergence was observed in L domains binding Alix, with the identification of three sequences: YPDL in equine infectious anemia virus (EIAV) (23, 26, 29), LYPLASLRSLF in human immunodeficiency virus type 1 (HIV-1), and the recently described SREKPYKEVTEDLLHLNSLF sequence in the simian immunodeficiency virus SIVmac239 (30). They have been dubbed types 1, 2, and 3, respectively. The last type contains an anchoring tyrosine, followed by one or two hydrophobic residues (valine and/or leucine) (proximal leucines), and both residues are key functional determinants for Alix (30). We observed that the Alix-binding type 3 L domain also contains a downstream LXXLF sequence carrying additional leucines (distal leucines) that are present in SIVmac239, SIVagmTan-1, and SIVsmE543. Similarly, the HIV-1 p6 domain also harbors an LXXLF motif in its LYPLASLRSLF L domain (underlined), which is critical for Alix binding and function (23, 26). In this study, we examined the role of distal leucine residues in the LNSLF sequence within the Alix-binding type 3 L domains of both SIVsmE543 (9) and SIVmac239 (12).
L domain function has recently been assessed in simian retroviruses and has led to the discovery of a new Alix-binding L domain in SIVmac239 (30). We examined the L domain function in SIVsmE543 (9). The Gag protein of this virus contains a total of two PTAP L domains located in the SIVsmE543 MA and p6 domains, respectively. Substitution of LTAA for the PTAP motif in p6 (smL2 mutant) eliminated ~50% of virus release from 293T (Fig. 1A, lane 3) and CEM cells (data not shown), whereas substitutions in MA had little effect on release (smL1 mutant), indicating that the p6 motif plays a dominant role in virus release. SIVsmE543 devoid of both motifs (smL1L2 mutant) retained ~50% of wild-type (WT) virus release (Fig. 1A, lane 4), suggesting that it harbors an additional L domain sequence(s). We searched for an Alix-binding L domain in the C-terminal region of the SIVsmE543 p6 domain and found a 488PYKEVTEDLLHLNSLF503L sequence, which is similar to the recently described Alix-binding type 3 L domain in SIVmac239 (Fig. 1B). To investigate the function of this motif, we replaced distal leucines 499L, 502L, and 503F in the 499LNSLF503 motif with prolines and a serine (smLL mutant) (Fig. 1B) and examined the effect of such changes. As expected, an smLL mutation in the context of WT SIVsmE543 had only a minimal affect on virus release (Fig. 1C, lane 2). Conversely, when the LL mutation was combined with L1 and L2 mutations (smL1L2/LL), virus release was obliterated (Fig. 1C, lanes 6 and 8). The effect of these mutations was comparable to that seen following the substitution of serines for the 489Y and 496L residues in the type 3 L domain (Fig. 1D, lanes 6 and 8), thus underscoring the importance of distal leucines in virus release. Similar results were obtained when distal leucines were mutated in the SIVmac239 LNSLF motif (macL2/LL and macL1L2/LL mutants) (Fig. 1E). These results, which were observed in three independent experiments (see quantification panels under the blots), indicate that distal leucines are an active part of the type 3 L domain and critical for Alix-dependent SIVsmE543 and SIVmac239 release.
Next, we assessed whether the 499L and LF503 residues are involved in Gag interactions with Alix. Cell lysates from 293T cells expressing SIVsmE543 smL1, smL2, and smLL mutants were tested for their ability to interact in immunoprecipitation assays with Flag-tagged Alix in the presence of 1% NP-40 detergent. Similar to observations with WT SIVsmE543 Gag, the smL1 and smL2 mutants were captured by Flag-Alix proteins (Fig. 2A, lanes 2, 4, and 6). In contrast, SIVsmE543 and SIVmac239 LL mutants (smLL and macLL) were not captured (Fig. 2A, lane 8, and C, lane 4). Likewise, substitutions of alanines for 489Y and 496L residues within SIVsmE543 and SIVmac239 (smYL and macYL mutants) eliminated binding to Alix (Fig. 2B, lane 4) (30). These results indicate that the Y and L residues are as critical as the distal leucines for the SIVsmE543 and SIVmac239 type 3 L domain binding to Alix.
To examine whether distal leucines 499L and 502LF503 are involved in Alix-mediated virus release, we used a virus rescue assay to test the effect of Flag-tagged Alix overexpression (22) on the release of a SIVsmE543 mutant that lacked the PTAP motif activity. Alix overexpression enhanced the release of SIVsmE543 lacking either the p6-located dominant PTAP motif (smL2 mutant) or both PTAP motifs in Gag (smL1L2 mutant) (Fig. 3A, lanes 2 and 6). Virus release augmentation was inhibited by the mutation of 499L and 502LF503 residues in the 499LNSLF503 motif (smL2/LL and smL1L2/LL mutants) (Fig. 3A, lanes 4 and 8) in a manner similar to that seen following the substitution of serines for 489Y and 496L (Fig. 3B). A similar result was obtained when the LNSLF motif was disrupted in SIVmac239 (Fig. 3C). These results were confirmed in three independent experiments, as shown in the release quantification panel in Fig. 3A. Together, they indicate that SIVsmE543 and SIVmac239 are less responsive to Alix overexpression when distal leucines 499L and 502LF503 are mutated.
When both PTAP L domains were disrupted in SIVsmE543 (smL1L2 mutant), ~50% of virus release was retained, as long as the Gag possessed an intact 488PYKEVTEDLLHLNSLF motif (Fig. 1 and and3).3). These findings suggest that the SIVsmE543 Gag utilizes the type 3 L domain sequence to recruit the host cell Alix and promote virus release. To examine whether distal leucines in the 499LNSLF motif are involved in the utilization of the host Alix, we knocked down Alix using RNAi and examined its effect on the WT or mutant viruses carrying either one (smL1 and smL2 mutants) or no (smL1L2 mutant) PTAP motif. As expected, RNAi depletion of cellular Alix had only a modest effect on the release of WT or L1 mutant viruses because of the second PTAP L domain dominance in the p6 region of Gag (Fig. 4A, lanes 3 and 6, and the release quantification below the blots). Conversely, depletion of cellular Alix decreased the release of SIVsmE543 L2 and L1L2 mutants to nearly undetectable levels (Fig. 4B, lanes 2 and 5), demonstrating that SIVsmE543 LNSLF is critical for the utilization of the host cell Alix during virus release.
Three types of Alix-binding L domains have been identified: YPDL, LYPLASLRSLF and SREKPYKEVTEDLLHLLNSLF motifs (key residues underlined) found within EIAV, HIV-1, and SIVmac239, respectively (23, 26, 30). These motifs share key binding and functional determinants. A tyrosine considered an “anchorage” of Alix (29, 30) is followed by hydrophobic residues, often leucines, which make contact with the Alix V domain (30). Here we report that two leucines and a phenylalanine residue in the SIVsmE543 488PYKEVTEDLLHLNSLF503 L domain (italics) play a key role in Alix function (Fig. 1 to to3),3), since substitutions at these residues resulted in loss of both binding and function. We conclude that in addition to the tyrosine and proximal hydrophobic residues in the type 3 L domain, additional relatively distant residues (distal leucines) are critical functional determinants of Alix. Interestingly, both leucines are found within an LXXLF sequence that is part of the type 3 L domains of SIVsmE543, SIVmac239, and SIVagmTAN-1 (30). An LXXLF motif is also found in HIV-1 and is critical for Alix recruitment and Alix-dependent release (23, 26). These observations confirm the conservation of the LXXLF sequence across primate lentiviruses, underscoring its importance in Alix-mediated virus release.
The Alix-binding type 3 L domain shares key structural and functional determinants with types 1 and 2, including the tyrosine (Y) and the hydrophobic proximal leucines that make contact within the V domain in order to stabilize binding with Alix (29, 30). Our data demonstrate that both functional determinants are required, but not sufficient, for the type 3 L domain binding to Alix, suggesting that further stabilization is necessary for recruitment and function. Consistent with this hypothesis, we found that leucines located 10 and 13 residues downstream of the tyrosine (distal leucines) in the PYKEVTEDLLHLNSLF sequence, are required for Alix binding to SIVsmE543 and SIVmac239 Gag proteins (Fig. 2). Similarly, leucines in the HIV-1 LYPLASLRSLF L domain (underlined) are also critical for Alix binding (17, 23, 26, 29). In contrast, distant leucines appear to be dispensable for the EIAV-borne type 1 L domain. One possible explanation for these discrepant requirements is the ability of the latter L domain to bind Alix with a high affinity (29). Together, these observations suggest that distal leucines in type 2 and 3 L domains are involved in further stabilization of Alix-p6 interactions, possibly to compensate for their low-affinity binding to p6.
Virus release was completely eliminated when mutation of distal leucines was combined with the disruption of both PTAP L domains in SIVsmE543. This suggested that the entire PYKEVTEDLLHLNSLF sequence functions as an additional L domain in p6 (Fig. 1). Such a notion is further supported by the reliance of the type 3 L domain on cellular Alix to drive virus release in the absence of PTAP motifs (Fig. 4). In summary, our data identify distal leucine residues in the SIVsmE543 and SIVmac239 type 3 Alix-binding L domains as key functional determinants.
We thank Alicia Buckler-White and her group for DNA sequencing and Ilnour Ourmanov for help with SIV protein detection.
This work was supported by the Intramural Research Program of the NIAID, NIH.
Published ahead of print on 17 August 2011.