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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

Mutation of potential trafficking motifs affects virion incorporation of SIVmac239 Env.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 SIVmac239 wild-type (WT) virions (R239). (C) Mutants YG, E4b, E6b and E7b were quantified in triplicate. Shown is the average of independent experiments, error bars indicate standard deviation.
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pone-0114753-g004: Mutation of potential trafficking motifs affects virion incorporation of SIVmac239 Env.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 SIVmac239 wild-type (WT) virions (R239). (C) Mutants YG, E4b, E6b and E7b were quantified in triplicate. Shown is the average of independent experiments, error bars indicate standard deviation.

Mentions: Next, we tested the extent to which mutation of the potential trafficking motifs in gp41CD affected incorporation of SIVmac239 Env into virions. To this end, infectious virus was produced in HEK293T cells and pelleted from clarified supernatant. Virion samples normalized for p27 content were then subjected to analysis by Western blot (Fig. 4A) and the ratio of Env to p27 in mutant virions relative to wild-type virions was determined by densitometry (Fig. 4B, C and Table 1). In stark contrast to HIV-1 Env, whose incorporation has been reported to be strongly reduced in mutants of potential gp41CD trafficking motifs [24], most mutant forms of SIV Env were incorporated into virions at higher levels than wild-type Env. Mutation of the membrane-proximal YXXΦ motif alone increased Env incorporation by 3.7-fold, as reported previously [23]. Disruption of the next eight potential trafficking motifs (mutants E1b – E4b), which cluster around the center of gp41CD, slightly enhanced Env incorporation further, with mutant E4b showing the highest level at 8.2-fold more Env than wild-type virions. Further mutations reduced Env incorporation in a step-wise fashion. Mutant E6b, which retains only the C-terminal dileucine motif, incorporated Env at levels comparable to wild-type virus, whereas mutant E7b, lacking all potential trafficking motifs, incorporated Env at merely 39% of wild-type virus. While not specifically included in parallel in these experiments, previous results from our laboratory have demonstrated a 25-fold increase in Env incorporation into SIVmac239 virions with the E767* truncation [23], [39].


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)

Mutation of potential trafficking motifs affects virion incorporation of SIVmac239 Env.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 SIVmac239 wild-type (WT) virions (R239). (C) Mutants YG, E4b, E6b and E7b were quantified in triplicate. Shown is the average of independent experiments, error bars indicate standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4257708&req=5

pone-0114753-g004: Mutation of potential trafficking motifs affects virion incorporation of SIVmac239 Env.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 SIVmac239 wild-type (WT) virions (R239). (C) Mutants YG, E4b, E6b and E7b were quantified in triplicate. Shown is the average of independent experiments, error bars indicate standard deviation.
Mentions: Next, we tested the extent to which mutation of the potential trafficking motifs in gp41CD affected incorporation of SIVmac239 Env into virions. To this end, infectious virus was produced in HEK293T cells and pelleted from clarified supernatant. Virion samples normalized for p27 content were then subjected to analysis by Western blot (Fig. 4A) and the ratio of Env to p27 in mutant virions relative to wild-type virions was determined by densitometry (Fig. 4B, C and Table 1). In stark contrast to HIV-1 Env, whose incorporation has been reported to be strongly reduced in mutants of potential gp41CD trafficking motifs [24], most mutant forms of SIV Env were incorporated into virions at higher levels than wild-type Env. Mutation of the membrane-proximal YXXΦ motif alone increased Env incorporation by 3.7-fold, as reported previously [23]. Disruption of the next eight potential trafficking motifs (mutants E1b – E4b), which cluster around the center of gp41CD, slightly enhanced Env incorporation further, with mutant E4b showing the highest level at 8.2-fold more Env than wild-type virions. Further mutations reduced Env incorporation in a step-wise fashion. Mutant E6b, which retains only the C-terminal dileucine motif, incorporated Env at levels comparable to wild-type virus, whereas mutant E7b, lacking all potential trafficking motifs, incorporated Env at merely 39% of wild-type virus. While not specifically included in parallel in these experiments, previous results from our laboratory have demonstrated a 25-fold increase in Env incorporation into SIVmac239 virions with the E767* truncation [23], [39].

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