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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

Point mutations of potential trafficking motifs do not affect cellular expression levels of SIVmac239 Env.HEK293T cells were transfected with SIVmac239 Env mutants. On day 3 after transfection, cells were lysed and equivalent amounts of lysate analyzed by Western blot. gp120 and gp160 were detected by antibody 3.11H. Tubulin was used as input control. WT, wild-type; E767*, Env mutant with truncation at amino acid E767.
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pone-0114753-g002: Point mutations of potential trafficking motifs do not affect cellular expression levels of SIVmac239 Env.HEK293T cells were transfected with SIVmac239 Env mutants. On day 3 after transfection, cells were lysed and equivalent amounts of lysate analyzed by Western blot. gp120 and gp160 were detected by antibody 3.11H. Tubulin was used as input control. WT, wild-type; E767*, Env mutant with truncation at amino acid E767.

Mentions: To test whether any of these mutations affected protein stability or Env cleavage, all nine mutant proteins were transiently expressed in HEK293T cells by DNA transfection and lysates were analyzed by Western blot. We used the Env expression cassette pSIVΔgpV [34], which is derived from the full-length molecular clone of SIVmac239 with major deletions in the gag-pol region and expresses Env under control of the LTR promoter, rather than a highly expressing codon-optimized Env plasmid, to avoid possible complications from non-physiologically high levels of Env produced by the transfected cell or from the absence of Nef, which has been shown to interact with the trafficking machinery of the host cell [40]. Expression levels of all Env variants with amino-acid substitutions were comparable to the wild-type protein and cleavage of gp160 into gp120 and gp41 was not impaired (Fig. 2). Intriguingly, the truncated Env mutant E767* exhibited much higher levels of protein in whole-cell lysates, indicating that removing the C-terminal 113 amino acids from gp41CD drastically enhances protein expression and/or stability.


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)

Point mutations of potential trafficking motifs do not affect cellular expression levels of SIVmac239 Env.HEK293T cells were transfected with SIVmac239 Env mutants. On day 3 after transfection, cells were lysed and equivalent amounts of lysate analyzed by Western blot. gp120 and gp160 were detected by antibody 3.11H. Tubulin was used as input control. WT, wild-type; E767*, Env mutant with truncation at amino acid E767.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114753-g002: Point mutations of potential trafficking motifs do not affect cellular expression levels of SIVmac239 Env.HEK293T cells were transfected with SIVmac239 Env mutants. On day 3 after transfection, cells were lysed and equivalent amounts of lysate analyzed by Western blot. gp120 and gp160 were detected by antibody 3.11H. Tubulin was used as input control. WT, wild-type; E767*, Env mutant with truncation at amino acid E767.
Mentions: To test whether any of these mutations affected protein stability or Env cleavage, all nine mutant proteins were transiently expressed in HEK293T cells by DNA transfection and lysates were analyzed by Western blot. We used the Env expression cassette pSIVΔgpV [34], which is derived from the full-length molecular clone of SIVmac239 with major deletions in the gag-pol region and expresses Env under control of the LTR promoter, rather than a highly expressing codon-optimized Env plasmid, to avoid possible complications from non-physiologically high levels of Env produced by the transfected cell or from the absence of Nef, which has been shown to interact with the trafficking machinery of the host cell [40]. Expression levels of all Env variants with amino-acid substitutions were comparable to the wild-type protein and cleavage of gp160 into gp120 and gp41 was not impaired (Fig. 2). Intriguingly, the truncated Env mutant E767* exhibited much higher levels of protein in whole-cell lysates, indicating that removing the C-terminal 113 amino acids from gp41CD drastically enhances protein expression and/or stability.

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