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Systematic analysis of intracellular trafficking motifs located within the cytoplasmic domain of simian immunodeficiency virus glycoprotein gp41.

Postler TS, Bixby JG, Desrosiers RC, Yuste E - PLoS ONE (2014)

Bottom Line: These minor effects of mutations in trafficking motifs form a stark contrast to the strong increases in cell-surface expression and Env incorporation which have previously been reported for large truncations of gp41CD.Surprisingly, mutation of potential trafficking motifs in gp41CD of SIVmac316, which differs by only one residue from gp41CD of SIVmac239, effectively recapitulated the increases in Env incorporation into virions observed with gp41CD truncations.Our results indicate that increases in Env surface expression and virion incorporation associated with truncation of SIVmac239 gp41CD are not fully explained by loss of consensus trafficking motifs.

View Article: PubMed Central - PubMed

Affiliation: New England Primate Research Center, Department of Microbiology and Immunobiology, Harvard Medical School, Southborough, Massachusetts, United States of America.

ABSTRACT
Previous studies have shown that truncation of the cytoplasmic-domain sequences of the simian immunodeficiency virus (SIV) envelope glycoprotein (Env) just prior to a potential intracellular-trafficking signal of the sequence YIHF can strongly increase Env protein expression on the cell surface, Env incorporation into virions and, at least in some contexts, virion infectivity. Here, all 12 potential intracellular-trafficking motifs (YXXΦ or LL/LI/IL) in the gp41 cytoplasmic domain (gp41CD) of SIVmac239 were analyzed by systematic mutagenesis. One single and 7 sequential combination mutants in this cytoplasmic domain were characterized. Cell-surface levels of Env were not significantly affected by any of the mutations. Most combination mutations resulted in moderate 3- to 8-fold increases in Env incorporation into virions. However, mutation of all 12 potential sites actually decreased Env incorporation into virions. Variant forms with 11 or 12 mutated sites exhibited 3-fold lower levels of inherent infectivity, while none of the other single or combination mutations that were studied significantly affected the inherent infectivity of SIVmac239. These minor effects of mutations in trafficking motifs form a stark contrast to the strong increases in cell-surface expression and Env incorporation which have previously been reported for large truncations of gp41CD. Surprisingly, mutation of potential trafficking motifs in gp41CD of SIVmac316, which differs by only one residue from gp41CD of SIVmac239, effectively recapitulated the increases in Env incorporation into virions observed with gp41CD truncations. Our results indicate that increases in Env surface expression and virion incorporation associated with truncation of SIVmac239 gp41CD are not fully explained by loss of consensus trafficking motifs.

No MeSH data available.


Related in: MedlinePlus

Virion incorporation of SIVmac316 Env is highly responsive to mutations of potential trafficking motifs.Viruses were produced by transfection of HEK293T cells and virions were pelleted from the clarified supernatants. (A) gp160/gp120 (top panel) and p27 (bottom panel) were detected by Western blot using antibody 3.11H and 2F12, respectively. (B) Relative Env incorporation into virions. The ratios of gp120 to p27 were calculated by phosphorimaging analysis. Data are presented as the gp120:p27 ratio of mutant virions (Rn) relative to the gp120:p27 ratio of SIVmac316 wild-type (WT) virions (R316). Data shown are representative of three independent experiments.
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pone-0114753-g007: Virion incorporation of SIVmac316 Env is highly responsive to mutations of potential trafficking motifs.Viruses were produced by transfection of HEK293T cells and virions were pelleted from the clarified supernatants. (A) gp160/gp120 (top panel) and p27 (bottom panel) were detected by Western blot using antibody 3.11H and 2F12, respectively. (B) Relative Env incorporation into virions. The ratios of gp120 to p27 were calculated by phosphorimaging analysis. Data are presented as the gp120:p27 ratio of mutant virions (Rn) relative to the gp120:p27 ratio of SIVmac316 wild-type (WT) virions (R316). Data shown are representative of three independent experiments.

Mentions: Env encoded by SIVmac316 (Env316) differs from Env of SIVmac239 (Env239) by only eight amino acids, one of which is located within gp41CD, but has been shown to differ considerably with respect to multiple structural and functional properties [31], [36], [42]. In particular, Env316 has been postulated to have a more “open” conformation than its SIVmac239 counterpart resulting in increased neutralization sensitivity [36]. Moreover, truncation of Env316 at amino acid E767 has been shown to increase Env incorporation into virions similar to or slightly less than truncation of Env239; however, the increase in viral infectivity of SIVmac316 resulting from this mutation has been reported as an exceptional 480-fold relative to the wild-type parent strain, compared to a modest 2.5-fold increase for SIVmac239 [39]. Importantly, virion incorporation of Env316 is only half as efficient as Env239 incorporation, and the inherent infectivity of SIVmac316 is about 20-fold lower than the inherent infectivity of SIVmac239 [39]. To gauge the importance of the potential trafficking motifs in gp41CD in the context of an altered structure with similar amino-acid sequence, mutations YG, E3b and E7b (Fig. 1) were introduced into the genetic background of SIVmac316. Virions were produced by transfection of HEK293T cells, pelleted and p27-normalized samples analyzed by Western blot to measure incorporation of Env into virions (Fig. 7). Removal of only the membrane-proximal YXXΦ motif was sufficient to strongly increase Env incorporation into SIVmac316 virions. Interestingly, mutant Env316 YG also incorporated a disproportionately high amount of gp160 into virions, which was not observed with other mutants. Disruption of the first six potential trafficking motifs in gp41CD (mutant E3b) increased Env316 incorporation even more strongly. Consistent with the notion that the C-terminal trafficking motifs may be required for efficient Env incorporation, Env316 mutant E7b was included in virions far less effectively than mutants YG or E3b, but still at higher levels than wild-type Env316. These data further substantiate the observation that the membrane-proximal YXXΦ motif and the central trafficking motifs serve to reduce the level of Env incorporation into virions, whereas the C-terminal motifs seem to contribute to incorporation. It is important to note that, while mutations in Env239 and Env316 affected virion incorporation in a qualitatively similar fashion, Env316 appeared to be far more responsive to mutations of potential trafficking motifs in gp41CD than Env239 and essentially recapitulated the phenotype observed with the E767* mutant (Table 2).


Systematic analysis of intracellular trafficking motifs located within the cytoplasmic domain of simian immunodeficiency virus glycoprotein gp41.

Postler TS, Bixby JG, Desrosiers RC, Yuste E - PLoS ONE (2014)

Virion incorporation of SIVmac316 Env is highly responsive to mutations of potential trafficking motifs.Viruses were produced by transfection of HEK293T cells and virions were pelleted from the clarified supernatants. (A) gp160/gp120 (top panel) and p27 (bottom panel) were detected by Western blot using antibody 3.11H and 2F12, respectively. (B) Relative Env incorporation into virions. The ratios of gp120 to p27 were calculated by phosphorimaging analysis. Data are presented as the gp120:p27 ratio of mutant virions (Rn) relative to the gp120:p27 ratio of SIVmac316 wild-type (WT) virions (R316). Data shown are representative of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4257708&req=5

pone-0114753-g007: Virion incorporation of SIVmac316 Env is highly responsive to mutations of potential trafficking motifs.Viruses were produced by transfection of HEK293T cells and virions were pelleted from the clarified supernatants. (A) gp160/gp120 (top panel) and p27 (bottom panel) were detected by Western blot using antibody 3.11H and 2F12, respectively. (B) Relative Env incorporation into virions. The ratios of gp120 to p27 were calculated by phosphorimaging analysis. Data are presented as the gp120:p27 ratio of mutant virions (Rn) relative to the gp120:p27 ratio of SIVmac316 wild-type (WT) virions (R316). Data shown are representative of three independent experiments.
Mentions: Env encoded by SIVmac316 (Env316) differs from Env of SIVmac239 (Env239) by only eight amino acids, one of which is located within gp41CD, but has been shown to differ considerably with respect to multiple structural and functional properties [31], [36], [42]. In particular, Env316 has been postulated to have a more “open” conformation than its SIVmac239 counterpart resulting in increased neutralization sensitivity [36]. Moreover, truncation of Env316 at amino acid E767 has been shown to increase Env incorporation into virions similar to or slightly less than truncation of Env239; however, the increase in viral infectivity of SIVmac316 resulting from this mutation has been reported as an exceptional 480-fold relative to the wild-type parent strain, compared to a modest 2.5-fold increase for SIVmac239 [39]. Importantly, virion incorporation of Env316 is only half as efficient as Env239 incorporation, and the inherent infectivity of SIVmac316 is about 20-fold lower than the inherent infectivity of SIVmac239 [39]. To gauge the importance of the potential trafficking motifs in gp41CD in the context of an altered structure with similar amino-acid sequence, mutations YG, E3b and E7b (Fig. 1) were introduced into the genetic background of SIVmac316. Virions were produced by transfection of HEK293T cells, pelleted and p27-normalized samples analyzed by Western blot to measure incorporation of Env into virions (Fig. 7). Removal of only the membrane-proximal YXXΦ motif was sufficient to strongly increase Env incorporation into SIVmac316 virions. Interestingly, mutant Env316 YG also incorporated a disproportionately high amount of gp160 into virions, which was not observed with other mutants. Disruption of the first six potential trafficking motifs in gp41CD (mutant E3b) increased Env316 incorporation even more strongly. Consistent with the notion that the C-terminal trafficking motifs may be required for efficient Env incorporation, Env316 mutant E7b was included in virions far less effectively than mutants YG or E3b, but still at higher levels than wild-type Env316. These data further substantiate the observation that the membrane-proximal YXXΦ motif and the central trafficking motifs serve to reduce the level of Env incorporation into virions, whereas the C-terminal motifs seem to contribute to incorporation. It is important to note that, while mutations in Env239 and Env316 affected virion incorporation in a qualitatively similar fashion, Env316 appeared to be far more responsive to mutations of potential trafficking motifs in gp41CD than Env239 and essentially recapitulated the phenotype observed with the E767* mutant (Table 2).

Bottom Line: These minor effects of mutations in trafficking motifs form a stark contrast to the strong increases in cell-surface expression and Env incorporation which have previously been reported for large truncations of gp41CD.Surprisingly, mutation of potential trafficking motifs in gp41CD of SIVmac316, which differs by only one residue from gp41CD of SIVmac239, effectively recapitulated the increases in Env incorporation into virions observed with gp41CD truncations.Our results indicate that increases in Env surface expression and virion incorporation associated with truncation of SIVmac239 gp41CD are not fully explained by loss of consensus trafficking motifs.

View Article: PubMed Central - PubMed

Affiliation: New England Primate Research Center, Department of Microbiology and Immunobiology, Harvard Medical School, Southborough, Massachusetts, United States of America.

ABSTRACT
Previous studies have shown that truncation of the cytoplasmic-domain sequences of the simian immunodeficiency virus (SIV) envelope glycoprotein (Env) just prior to a potential intracellular-trafficking signal of the sequence YIHF can strongly increase Env protein expression on the cell surface, Env incorporation into virions and, at least in some contexts, virion infectivity. Here, all 12 potential intracellular-trafficking motifs (YXXΦ or LL/LI/IL) in the gp41 cytoplasmic domain (gp41CD) of SIVmac239 were analyzed by systematic mutagenesis. One single and 7 sequential combination mutants in this cytoplasmic domain were characterized. Cell-surface levels of Env were not significantly affected by any of the mutations. Most combination mutations resulted in moderate 3- to 8-fold increases in Env incorporation into virions. However, mutation of all 12 potential sites actually decreased Env incorporation into virions. Variant forms with 11 or 12 mutated sites exhibited 3-fold lower levels of inherent infectivity, while none of the other single or combination mutations that were studied significantly affected the inherent infectivity of SIVmac239. These minor effects of mutations in trafficking motifs form a stark contrast to the strong increases in cell-surface expression and Env incorporation which have previously been reported for large truncations of gp41CD. Surprisingly, mutation of potential trafficking motifs in gp41CD of SIVmac316, which differs by only one residue from gp41CD of SIVmac239, effectively recapitulated the increases in Env incorporation into virions observed with gp41CD truncations. Our results indicate that increases in Env surface expression and virion incorporation associated with truncation of SIVmac239 gp41CD are not fully explained by loss of consensus trafficking motifs.

No MeSH data available.


Related in: MedlinePlus