Limits...
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

Viral infectivity of SIVmac239 is not affected by mutation of most potential trafficking motifs in gp41CD.Viruses were produced by transfection of HEK293T cells and virions were pelleted from the clarified supernatants. Stocks were normalized for p27 content and used to infect C8166-45 SIV-SEAP cells with serial dilutions. (A) SEAP activity 3 days after infection. Error bars indicate standard deviation of triplicates. (B) SEAP activity per ng of p27 input, normalized for wild-type virus. Average of 2–3 independent experiments, error bars indicate standard deviation. WT, wild-type. SEAP, secreted alkaline phosphatase.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4257708&req=5

pone-0114753-g005: Viral infectivity of SIVmac239 is not affected by mutation of most potential trafficking motifs in gp41CD.Viruses were produced by transfection of HEK293T cells and virions were pelleted from the clarified supernatants. Stocks were normalized for p27 content and used to infect C8166-45 SIV-SEAP cells with serial dilutions. (A) SEAP activity 3 days after infection. Error bars indicate standard deviation of triplicates. (B) SEAP activity per ng of p27 input, normalized for wild-type virus. Average of 2–3 independent experiments, error bars indicate standard deviation. WT, wild-type. SEAP, secreted alkaline phosphatase.

Mentions: To assess the relevance of the potential trafficking motifs in gp41CD for viral infectivity, we infected C8166-45 SIV-SEAP cells with mutant virus produced in HEK293T cells and normalized for p27 content. C8166-45 SIV-SEAP cells express secreted alkaline phosphatase (SEAP) upon infection with SIV; SEAP levels in the supernatant can be conveniently detected by a chemiluminescence-based assay and are directly proportional to the amount of virus infecting the cells [35]. Mutation of most potential trafficking motifs in gp41CD did not have major effects on viral infectivity (Fig. 5 and Table 1, mutants YG and E1b – E5b). In a previous study, we have demonstrated that even a 25-fold increase in Env incorporation increases infectivity of SIVmac239 by only 2.5-fold [23]. Therefore, it is to be expected that the observed increase in Env incorporation of up to 8.2-fold (Fig. 4 and Table 1) would not translate into a drastic change in viral infectivity. Eliminating 11 or 12 of the motifs, however, reduced infectivity by 3-fold compared to wild-type virus (Fig. 5 and Table 1, mutants E6b and E7b). Interestingly, mutant E6b retained near-wild-type levels of Env incorporation into virions; thus, the reduction in viral infectivity cannot be attributed to a reduction in Env incorporation, but more likely results from some other structure/function defect.


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)

Viral infectivity of SIVmac239 is not affected by mutation of most potential trafficking motifs in gp41CD.Viruses were produced by transfection of HEK293T cells and virions were pelleted from the clarified supernatants. Stocks were normalized for p27 content and used to infect C8166-45 SIV-SEAP cells with serial dilutions. (A) SEAP activity 3 days after infection. Error bars indicate standard deviation of triplicates. (B) SEAP activity per ng of p27 input, normalized for wild-type virus. Average of 2–3 independent experiments, error bars indicate standard deviation. WT, wild-type. SEAP, secreted alkaline phosphatase.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114753-g005: Viral infectivity of SIVmac239 is not affected by mutation of most potential trafficking motifs in gp41CD.Viruses were produced by transfection of HEK293T cells and virions were pelleted from the clarified supernatants. Stocks were normalized for p27 content and used to infect C8166-45 SIV-SEAP cells with serial dilutions. (A) SEAP activity 3 days after infection. Error bars indicate standard deviation of triplicates. (B) SEAP activity per ng of p27 input, normalized for wild-type virus. Average of 2–3 independent experiments, error bars indicate standard deviation. WT, wild-type. SEAP, secreted alkaline phosphatase.
Mentions: To assess the relevance of the potential trafficking motifs in gp41CD for viral infectivity, we infected C8166-45 SIV-SEAP cells with mutant virus produced in HEK293T cells and normalized for p27 content. C8166-45 SIV-SEAP cells express secreted alkaline phosphatase (SEAP) upon infection with SIV; SEAP levels in the supernatant can be conveniently detected by a chemiluminescence-based assay and are directly proportional to the amount of virus infecting the cells [35]. Mutation of most potential trafficking motifs in gp41CD did not have major effects on viral infectivity (Fig. 5 and Table 1, mutants YG and E1b – E5b). In a previous study, we have demonstrated that even a 25-fold increase in Env incorporation increases infectivity of SIVmac239 by only 2.5-fold [23]. Therefore, it is to be expected that the observed increase in Env incorporation of up to 8.2-fold (Fig. 4 and Table 1) would not translate into a drastic change in viral infectivity. Eliminating 11 or 12 of the motifs, however, reduced infectivity by 3-fold compared to wild-type virus (Fig. 5 and Table 1, mutants E6b and E7b). Interestingly, mutant E6b retained near-wild-type levels of Env incorporation into virions; thus, the reduction in viral infectivity cannot be attributed to a reduction in Env incorporation, but more likely results from some other structure/function defect.

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