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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 cell cocultures after infection by G367R pseudotyped with VSV-G viruses. MT2 or MT4 cells were infected for 3 hours at 37°C, then washed with medium, and 105 cells were split into 2 wells for growth assays. 105 infected MT4 cells after 7 days from each well were split into 2 wells and mixed with fresh 2×105 MT2 cells. Two curves are shown separately because the values of the duplicate cocultures were different.
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Fig3: p24 production in MT2-MT4 cell cocultures after infection by G367R pseudotyped with VSV-G viruses. MT2 or MT4 cells were infected for 3 hours at 37°C, then washed with medium, and 105 cells were split into 2 wells for growth assays. 105 infected MT4 cells after 7 days from each well were split into 2 wells and mixed with fresh 2×105 MT2 cells. Two curves are shown separately because the values of the duplicate cocultures were different.

Mentions: Next, we investigated whether donor or target cells determine the efficiency of reversion of the G367R mutant. As shown above, coculturing G367R positive 293 T and TZM-bl cells, but not their supernatants, with MT2 or MT4 cells confirmed that the cell-associated the G367R virus can infect MT2 cells and revert to WT but cannot infect MT4 cells (Table 4). This result indicates that the type of target cell is important in determining the reversion of G367R mutant. The fact that MT4 cells infected by diluted G367R viruses that were pseudotyped with VSV-G did not revert is also in agreement with this result (Table 2). This finding also indicates that the mutant G367R HIV-1 cannot transmit if MT4 cells serve as both donor and target cells, as p24 values decreased gradually (Figure 3). In contrast, MT4 donor cells can transmit HIV-1 G367R mutant to MT2 target cells. In our experiments, CPE appeared after about 3 weeks in cocultures of MT4 cells containing G367R provirus, i.e., MT4 donor cells with uninfected MT2 cells. Reversion of phenotype was confirmed by a steep increase in p24 values in culture fluids (Figure 3) and the ability of the supernatants to participate in a second round of infection via cell-free transmission (data not shown). Thus, the G367R mutant has no deficit in ability to bud from MT4 cells. Moreover, it can be concluded that the efficiency of the G367R reversion is completely determined by the type of target cell used and not by donor cells.Figure 3


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 cell cocultures after infection by G367R pseudotyped with VSV-G viruses. MT2 or MT4 cells were infected for 3 hours at 37°C, then washed with medium, and 105 cells were split into 2 wells for growth assays. 105 infected MT4 cells after 7 days from each well were split into 2 wells and mixed with fresh 2×105 MT2 cells. Two curves are shown separately because the values of the duplicate cocultures were different.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: p24 production in MT2-MT4 cell cocultures after infection by G367R pseudotyped with VSV-G viruses. MT2 or MT4 cells were infected for 3 hours at 37°C, then washed with medium, and 105 cells were split into 2 wells for growth assays. 105 infected MT4 cells after 7 days from each well were split into 2 wells and mixed with fresh 2×105 MT2 cells. Two curves are shown separately because the values of the duplicate cocultures were different.
Mentions: Next, we investigated whether donor or target cells determine the efficiency of reversion of the G367R mutant. As shown above, coculturing G367R positive 293 T and TZM-bl cells, but not their supernatants, with MT2 or MT4 cells confirmed that the cell-associated the G367R virus can infect MT2 cells and revert to WT but cannot infect MT4 cells (Table 4). This result indicates that the type of target cell is important in determining the reversion of G367R mutant. The fact that MT4 cells infected by diluted G367R viruses that were pseudotyped with VSV-G did not revert is also in agreement with this result (Table 2). This finding also indicates that the mutant G367R HIV-1 cannot transmit if MT4 cells serve as both donor and target cells, as p24 values decreased gradually (Figure 3). In contrast, MT4 donor cells can transmit HIV-1 G367R mutant to MT2 target cells. In our experiments, CPE appeared after about 3 weeks in cocultures of MT4 cells containing G367R provirus, i.e., MT4 donor cells with uninfected MT2 cells. Reversion of phenotype was confirmed by a steep increase in p24 values in culture fluids (Figure 3) and the ability of the supernatants to participate in a second round of infection via cell-free transmission (data not shown). Thus, the G367R mutant has no deficit in ability to bud from MT4 cells. Moreover, it can be concluded that the efficiency of the G367R reversion is completely determined by the type of target cell used and not by donor cells.Figure 3

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