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Identification of an env-defective HIV-1 mutant capable of spontaneous reversion to a wild-type phenotype in certain T-cell lines.

Quan Y, Xu H, Kramer VG, Han Y, Sloan RD, Wainberg MA - Virol. J. (2014)

Bottom Line: Mutations in the env gene of HIV may be able to exert a differential influence on viral transmission ability in regard to cell-free and cell-associated viral forms.The persistence of defective forms can potentially lead to the emergence of virulent forms.The heterogeneity of cell types that constitute the HIV reservoir can contribute to viral variability, even among similar types of cells.

View Article: PubMed Central - PubMed

Affiliation: McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Cote Sainte Catherine, Montreal, QC H3T 1E2, Canada. mark.wainberg@mcgill.ca.

ABSTRACT

Background: Attempts to eradicate HIV from cellular reservoirs are vital but depend on a clear understanding of how viral variants are transmitted and survive in the different cell types that constitute such reservoirs. Mutations in the env gene of HIV may be able to exert a differential influence on viral transmission ability in regard to cell-free and cell-associated viral forms.

Methods: The ability of HIV containing an env G367R mutation in cell-free and cell-associated viruses to cause infection and to revert to wild-type was measured using several T cell lines. To determine factors that might potentially influence the reversion of G367R, we studied each of entry inhibitors, inhibitors of cellular endocytosis, and modulators of cell growth and activation.

Results: We demonstrate that an HIV-1 variant containing a G367R substitution within the CD4 binding site of gp120 was non-infectious as free virus in culture but was infectious when infected cells were co-cultured with certain T cell lines or when cells were transfected by a relevant proviral plasmid. Differences in viral infectivity by cell-associated G367R viruses were determined by the type of target cell employed, regardless which type of donor cell was used. Reversion was slowed or inhibited by entry inhibitors and by inhibitors of cellular endocytosis. Interleukin 2 was able to block G367R reversion in only one of the T cell lines studied but not in the other, while phorbol 12-myristate 13-acetate (PMA) inhibited G367R reversion in all the T cell lines.

Conclusions: Env-defective HIV may have a different phenotype as cell-free versus cell-associated virus. The persistence of defective forms can potentially lead to the emergence of virulent forms. The heterogeneity of cell types that constitute the HIV reservoir can contribute to viral variability, even among similar types of cells. This is the first demonstration of a mutation in the HIV envelope, i.e. G367R, that can compromise infection by cell-free virus but less severely by cell-associated virus and that does so in a cell type-dependent manner.

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p24 production in MT2, MT4 and SupT1 cells after infection by G367R virus that had been pseudotyped with VSV-G viruses in the presence of IL2, PMA or ionomycin. MT2 cells were infected for 3 hours at 37°C, then washed with medium, and 105 cells were split into wells in triplicate for growth in the presence or absence of 20 IU/ml of IL2 (a), 0.1, 0.5 ng/ml of PMA or 25 ng/ml of ionomycin (b).
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Fig6: p24 production in MT2, MT4 and SupT1 cells after infection by G367R virus that had been pseudotyped with VSV-G viruses in the presence of IL2, PMA or ionomycin. MT2 cells were infected for 3 hours at 37°C, then washed with medium, and 105 cells were split into wells in triplicate for growth in the presence or absence of 20 IU/ml of IL2 (a), 0.1, 0.5 ng/ml of PMA or 25 ng/ml of ionomycin (b).

Mentions: Based on the above results, G367R reversion may be determined by undefined cellular mechanisms. Contrary to expectations, we found that IL2 was able to inhibit the reversion of G367R virus in MT2 cells at the same concentration used to maintain T lymphocyte growth (Figure 6a) without affecting cell viability, while it only slightly inhibited cell-free wt HIV by about 2 fold (data not shown). In contrast, IL2 had no influence on the reversion of G367R in SupT1 cells, based on both p24 measurements and CPE (Figure 6a). Again, there was no growth or reversion of G367R in MT4 cells, suggesting that a different mechanism of reversion of G367R may be involved in different cells.However, PMA and ionomycin inhibited the reversion of G367R virus in both MT2 and SupT1 cells (Figure 6b). A low concentration of PMA of 0.1 ng/ml or ionomycin at 25 ng/ml both inhibited the G367R reversion. These results suggest that the reversion of G367R that is dependent on transmission between cells may be protein kinase C (PKC) related.Figure 6


Identification of an env-defective HIV-1 mutant capable of spontaneous reversion to a wild-type phenotype in certain T-cell lines.

Quan Y, Xu H, Kramer VG, Han Y, Sloan RD, Wainberg MA - Virol. J. (2014)

p24 production in MT2, MT4 and SupT1 cells after infection by G367R virus that had been pseudotyped with VSV-G viruses in the presence of IL2, PMA or ionomycin. MT2 cells were infected for 3 hours at 37°C, then washed with medium, and 105 cells were split into wells in triplicate for growth in the presence or absence of 20 IU/ml of IL2 (a), 0.1, 0.5 ng/ml of PMA or 25 ng/ml of ionomycin (b).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4283149&req=5

Fig6: p24 production in MT2, MT4 and SupT1 cells after infection by G367R virus that had been pseudotyped with VSV-G viruses in the presence of IL2, PMA or ionomycin. MT2 cells were infected for 3 hours at 37°C, then washed with medium, and 105 cells were split into wells in triplicate for growth in the presence or absence of 20 IU/ml of IL2 (a), 0.1, 0.5 ng/ml of PMA or 25 ng/ml of ionomycin (b).
Mentions: Based on the above results, G367R reversion may be determined by undefined cellular mechanisms. Contrary to expectations, we found that IL2 was able to inhibit the reversion of G367R virus in MT2 cells at the same concentration used to maintain T lymphocyte growth (Figure 6a) without affecting cell viability, while it only slightly inhibited cell-free wt HIV by about 2 fold (data not shown). In contrast, IL2 had no influence on the reversion of G367R in SupT1 cells, based on both p24 measurements and CPE (Figure 6a). Again, there was no growth or reversion of G367R in MT4 cells, suggesting that a different mechanism of reversion of G367R may be involved in different cells.However, PMA and ionomycin inhibited the reversion of G367R virus in both MT2 and SupT1 cells (Figure 6b). A low concentration of PMA of 0.1 ng/ml or ionomycin at 25 ng/ml both inhibited the G367R reversion. These results suggest that the reversion of G367R that is dependent on transmission between cells may be protein kinase C (PKC) related.Figure 6

Bottom Line: Mutations in the env gene of HIV may be able to exert a differential influence on viral transmission ability in regard to cell-free and cell-associated viral forms.The persistence of defective forms can potentially lead to the emergence of virulent forms.The heterogeneity of cell types that constitute the HIV reservoir can contribute to viral variability, even among similar types of cells.

View Article: PubMed Central - PubMed

Affiliation: McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Cote Sainte Catherine, Montreal, QC H3T 1E2, Canada. mark.wainberg@mcgill.ca.

ABSTRACT

Background: Attempts to eradicate HIV from cellular reservoirs are vital but depend on a clear understanding of how viral variants are transmitted and survive in the different cell types that constitute such reservoirs. Mutations in the env gene of HIV may be able to exert a differential influence on viral transmission ability in regard to cell-free and cell-associated viral forms.

Methods: The ability of HIV containing an env G367R mutation in cell-free and cell-associated viruses to cause infection and to revert to wild-type was measured using several T cell lines. To determine factors that might potentially influence the reversion of G367R, we studied each of entry inhibitors, inhibitors of cellular endocytosis, and modulators of cell growth and activation.

Results: We demonstrate that an HIV-1 variant containing a G367R substitution within the CD4 binding site of gp120 was non-infectious as free virus in culture but was infectious when infected cells were co-cultured with certain T cell lines or when cells were transfected by a relevant proviral plasmid. Differences in viral infectivity by cell-associated G367R viruses were determined by the type of target cell employed, regardless which type of donor cell was used. Reversion was slowed or inhibited by entry inhibitors and by inhibitors of cellular endocytosis. Interleukin 2 was able to block G367R reversion in only one of the T cell lines studied but not in the other, while phorbol 12-myristate 13-acetate (PMA) inhibited G367R reversion in all the T cell lines.

Conclusions: Env-defective HIV may have a different phenotype as cell-free versus cell-associated virus. The persistence of defective forms can potentially lead to the emergence of virulent forms. The heterogeneity of cell types that constitute the HIV reservoir can contribute to viral variability, even among similar types of cells. This is the first demonstration of a mutation in the HIV envelope, i.e. G367R, that can compromise infection by cell-free virus but less severely by cell-associated virus and that does so in a cell type-dependent manner.

Show MeSH
Related in: MedlinePlus