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Role of the C-terminal domain of the HIV-1 glycoprotein in cell-to-cell viral transmission between T lymphocytes.

Emerson V, Haller C, Pfeiffer T, Fackler OT, Bosch V - Retrovirology (2010)

Bottom Line: We further observed that in permissive cells, which supported both routes of mutant virus transmission, viral gene expression levels, Gag processing and particle release were inherently higher than in non-permissive cells, a factor which may be significantly contributing to their permissivity phenotype.Additionally, and correlating with viral transfer efficiencies in these cell types, HIV-Gag accumulation at the virological synapse (VS) was reduced to background levels in the absence of the Env-CT in conjugates of non-permissive cells but not in permissive cells.The requirement of the Env-CT for these related processes is abrogated in permissive cells, which exhibit higher HIV gene expression levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Forschungsschwerpunkt Infektion und Krebs, F020, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

ABSTRACT

Background: Mutant HIV (HIV-Env-Tr712) lacking the cytoplasmic tail of the viral glycoprotein (Env-CT) exhibits a cell-type specific replication phenotype such that replicative spread occurs in some T-cell lines (referred to as permissive cells) but fails to do so in most T-cell lines or in PBMCs (referred to as non-permissive cells). We aim to gain insight on the underlying requirement for the Env-CT for viral spread in non-permissive cells.

Results: We established that in comparison to HIV-Wt, both cell-free and cell-to-cell transmission of mutant HIV-Env-Tr712 from non-permissive cells were severely impaired under naturally low infection conditions. This requirement for Env-CT could be largely overcome by using saturating amounts of virus for infection. We further observed that in permissive cells, which supported both routes of mutant virus transmission, viral gene expression levels, Gag processing and particle release were inherently higher than in non-permissive cells, a factor which may be significantly contributing to their permissivity phenotype. Additionally, and correlating with viral transfer efficiencies in these cell types, HIV-Gag accumulation at the virological synapse (VS) was reduced to background levels in the absence of the Env-CT in conjugates of non-permissive cells but not in permissive cells.

Conclusions: During natural infection conditions, the HIV-Env-CT is critically required for viral transmission in cultures of non-permissive cells by both cell-free and cell-to-cell routes and is instrumental for Gag accumulation to the VS. The requirement of the Env-CT for these related processes is abrogated in permissive cells, which exhibit higher HIV gene expression levels.

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Analysis of CA accumulation at the VS. Confocal microscopic analysis of CA and F-actin localisation phenotypes in conjugates of non-permissive H9 cells or permissive MT-4 cells infected with HIV-Wt, HIV-Env-Tr712 or HIV-ΔEnv. Conjugates between infected donor cells and freshly added dye-labelled target cells were generated as described in the Material and Methods section and selected for analysis initially by widefield microscopy. A. Predominant CA localisation patterns in conjugates of H9 (np) cells weakly infected with either HIV-Wt (distinct accumulation at the cell contact site), HIV-Env-Tr712 or HIV-ΔEnv (both diffuse cytoplasmic staining) as indicated. Dye-labelled cells, which were not visualised by our confocal microcope, are marked with × in the merge. B: Predominant CA localisation patterns in conjugates of weakly infected MT-4 (p) cells (CA accumulation at multiple sites at the cell periphery). Note that due to enhanced per cell CA expression levels in MT-4 cells, exposure times for taking the micrographs in B were approximately half as long as those employed in A in order to allow detection of individual CA clusters in both cases. C. The percentages of HIV-Wt conjugates exhibiting CA accumulation at the contact site (in the case of permissive MT-4 cells with or without accumulation elsewhere at the cell periphery) was set at 100% and the percentages of HIV-Env-Tr712 and HIV-ΔEnv conjugates exhibiting this phenotype calculated relative to this. The mean percentages from several experiments in weakly infected H9 cells (np, low) or MT-4 cells (p, low) are given.
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Figure 5: Analysis of CA accumulation at the VS. Confocal microscopic analysis of CA and F-actin localisation phenotypes in conjugates of non-permissive H9 cells or permissive MT-4 cells infected with HIV-Wt, HIV-Env-Tr712 or HIV-ΔEnv. Conjugates between infected donor cells and freshly added dye-labelled target cells were generated as described in the Material and Methods section and selected for analysis initially by widefield microscopy. A. Predominant CA localisation patterns in conjugates of H9 (np) cells weakly infected with either HIV-Wt (distinct accumulation at the cell contact site), HIV-Env-Tr712 or HIV-ΔEnv (both diffuse cytoplasmic staining) as indicated. Dye-labelled cells, which were not visualised by our confocal microcope, are marked with × in the merge. B: Predominant CA localisation patterns in conjugates of weakly infected MT-4 (p) cells (CA accumulation at multiple sites at the cell periphery). Note that due to enhanced per cell CA expression levels in MT-4 cells, exposure times for taking the micrographs in B were approximately half as long as those employed in A in order to allow detection of individual CA clusters in both cases. C. The percentages of HIV-Wt conjugates exhibiting CA accumulation at the contact site (in the case of permissive MT-4 cells with or without accumulation elsewhere at the cell periphery) was set at 100% and the percentages of HIV-Env-Tr712 and HIV-ΔEnv conjugates exhibiting this phenotype calculated relative to this. The mean percentages from several experiments in weakly infected H9 cells (np, low) or MT-4 cells (p, low) are given.

Mentions: It has been reported that during cell-to-cell HIV transmission, HIV-Gag protein accumulates at the VS and that this is reduced in the absence of Env [46]. Since the Env-CT has been shown to be able to influence Gag localisation in other cell systems [28,29], we were interested in comparing the localisation of HIV-Gag in Wt and mutant virion infected H9 (np) and MT-4 (p) cells. For this, cultures were weakly infected (to about 10%) with VSV-G pseudotyped HIV-Wt, HIV-Env-Tr712 or, as negative control, HIV-ΔEnv virions. 48 hours p.i., cell conjugates with non-infected cells were allowed to form and subsequently stained for HIV-CA and cellular F-actin. Analysis by confocal microscopy indicated that both in cultures of permissive or non-permissive cells, the absolute number of contacts was similar when using only uninfected cells as compared to mixtures of infected donors with uninfected targets. Furthermore, no major differences in the frequency of conjugation with uninfected target cells between HIV-Wt-, HIV-Env-Tr712- and HIV-ΔEnv-infected cells, irrespective of the permissivity status of the donor cell were observed (data not shown). This suggests that, at any rate in this cell system, conjugate formation is not necessarily driven by Env interaction with its cognate cellular receptor on the target cell. Polarisation of F-actin to cell-to-cell contacts was observed in some but not all cases, a variability that again did not correlate with cell permissivity or the Env variant used. In contrast, a large fraction (30-60% depending on the experiment) of H9 (np) cell conjugates infected with HIV-Wt displayed a marked accumulation of CA at the contact site (Fig. 5A, left panel) that was distinct from the diffuse cytoplasmic distribution observed in unconjugated cells (not shown). This result is in line with reports on the rapid polarization of the VS following contact formation between donor and target cell [7,14]. Notably, this polarisation of CA to cell-to-cell contacts was significantly reduced in conjugates with HIV-Env-Tr712- and HIV-ΔEnv-infected H9 cells and exhibited a similar diffuse cytoplasmic distribution as observed in unconjugated cells (Fig. 5A, middle and right panels). Quantification revealed that these reductions were to 43% and 51%, respectively, of HIV-Wt that was set to 100% (Fig. 5C, left panel). These findings presented here for HIV-1 are remarkably similar to a recent report on murine leukemia virus (MuLV) for which it has been demonstrated that polarised assembly at cell-cell contacts and release, but not cell conjugation, are mediated by the cytoplasmic tail of MuLV-Env [47].


Role of the C-terminal domain of the HIV-1 glycoprotein in cell-to-cell viral transmission between T lymphocytes.

Emerson V, Haller C, Pfeiffer T, Fackler OT, Bosch V - Retrovirology (2010)

Analysis of CA accumulation at the VS. Confocal microscopic analysis of CA and F-actin localisation phenotypes in conjugates of non-permissive H9 cells or permissive MT-4 cells infected with HIV-Wt, HIV-Env-Tr712 or HIV-ΔEnv. Conjugates between infected donor cells and freshly added dye-labelled target cells were generated as described in the Material and Methods section and selected for analysis initially by widefield microscopy. A. Predominant CA localisation patterns in conjugates of H9 (np) cells weakly infected with either HIV-Wt (distinct accumulation at the cell contact site), HIV-Env-Tr712 or HIV-ΔEnv (both diffuse cytoplasmic staining) as indicated. Dye-labelled cells, which were not visualised by our confocal microcope, are marked with × in the merge. B: Predominant CA localisation patterns in conjugates of weakly infected MT-4 (p) cells (CA accumulation at multiple sites at the cell periphery). Note that due to enhanced per cell CA expression levels in MT-4 cells, exposure times for taking the micrographs in B were approximately half as long as those employed in A in order to allow detection of individual CA clusters in both cases. C. The percentages of HIV-Wt conjugates exhibiting CA accumulation at the contact site (in the case of permissive MT-4 cells with or without accumulation elsewhere at the cell periphery) was set at 100% and the percentages of HIV-Env-Tr712 and HIV-ΔEnv conjugates exhibiting this phenotype calculated relative to this. The mean percentages from several experiments in weakly infected H9 cells (np, low) or MT-4 cells (p, low) are given.
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Figure 5: Analysis of CA accumulation at the VS. Confocal microscopic analysis of CA and F-actin localisation phenotypes in conjugates of non-permissive H9 cells or permissive MT-4 cells infected with HIV-Wt, HIV-Env-Tr712 or HIV-ΔEnv. Conjugates between infected donor cells and freshly added dye-labelled target cells were generated as described in the Material and Methods section and selected for analysis initially by widefield microscopy. A. Predominant CA localisation patterns in conjugates of H9 (np) cells weakly infected with either HIV-Wt (distinct accumulation at the cell contact site), HIV-Env-Tr712 or HIV-ΔEnv (both diffuse cytoplasmic staining) as indicated. Dye-labelled cells, which were not visualised by our confocal microcope, are marked with × in the merge. B: Predominant CA localisation patterns in conjugates of weakly infected MT-4 (p) cells (CA accumulation at multiple sites at the cell periphery). Note that due to enhanced per cell CA expression levels in MT-4 cells, exposure times for taking the micrographs in B were approximately half as long as those employed in A in order to allow detection of individual CA clusters in both cases. C. The percentages of HIV-Wt conjugates exhibiting CA accumulation at the contact site (in the case of permissive MT-4 cells with or without accumulation elsewhere at the cell periphery) was set at 100% and the percentages of HIV-Env-Tr712 and HIV-ΔEnv conjugates exhibiting this phenotype calculated relative to this. The mean percentages from several experiments in weakly infected H9 cells (np, low) or MT-4 cells (p, low) are given.
Mentions: It has been reported that during cell-to-cell HIV transmission, HIV-Gag protein accumulates at the VS and that this is reduced in the absence of Env [46]. Since the Env-CT has been shown to be able to influence Gag localisation in other cell systems [28,29], we were interested in comparing the localisation of HIV-Gag in Wt and mutant virion infected H9 (np) and MT-4 (p) cells. For this, cultures were weakly infected (to about 10%) with VSV-G pseudotyped HIV-Wt, HIV-Env-Tr712 or, as negative control, HIV-ΔEnv virions. 48 hours p.i., cell conjugates with non-infected cells were allowed to form and subsequently stained for HIV-CA and cellular F-actin. Analysis by confocal microscopy indicated that both in cultures of permissive or non-permissive cells, the absolute number of contacts was similar when using only uninfected cells as compared to mixtures of infected donors with uninfected targets. Furthermore, no major differences in the frequency of conjugation with uninfected target cells between HIV-Wt-, HIV-Env-Tr712- and HIV-ΔEnv-infected cells, irrespective of the permissivity status of the donor cell were observed (data not shown). This suggests that, at any rate in this cell system, conjugate formation is not necessarily driven by Env interaction with its cognate cellular receptor on the target cell. Polarisation of F-actin to cell-to-cell contacts was observed in some but not all cases, a variability that again did not correlate with cell permissivity or the Env variant used. In contrast, a large fraction (30-60% depending on the experiment) of H9 (np) cell conjugates infected with HIV-Wt displayed a marked accumulation of CA at the contact site (Fig. 5A, left panel) that was distinct from the diffuse cytoplasmic distribution observed in unconjugated cells (not shown). This result is in line with reports on the rapid polarization of the VS following contact formation between donor and target cell [7,14]. Notably, this polarisation of CA to cell-to-cell contacts was significantly reduced in conjugates with HIV-Env-Tr712- and HIV-ΔEnv-infected H9 cells and exhibited a similar diffuse cytoplasmic distribution as observed in unconjugated cells (Fig. 5A, middle and right panels). Quantification revealed that these reductions were to 43% and 51%, respectively, of HIV-Wt that was set to 100% (Fig. 5C, left panel). These findings presented here for HIV-1 are remarkably similar to a recent report on murine leukemia virus (MuLV) for which it has been demonstrated that polarised assembly at cell-cell contacts and release, but not cell conjugation, are mediated by the cytoplasmic tail of MuLV-Env [47].

Bottom Line: We further observed that in permissive cells, which supported both routes of mutant virus transmission, viral gene expression levels, Gag processing and particle release were inherently higher than in non-permissive cells, a factor which may be significantly contributing to their permissivity phenotype.Additionally, and correlating with viral transfer efficiencies in these cell types, HIV-Gag accumulation at the virological synapse (VS) was reduced to background levels in the absence of the Env-CT in conjugates of non-permissive cells but not in permissive cells.The requirement of the Env-CT for these related processes is abrogated in permissive cells, which exhibit higher HIV gene expression levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Forschungsschwerpunkt Infektion und Krebs, F020, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

ABSTRACT

Background: Mutant HIV (HIV-Env-Tr712) lacking the cytoplasmic tail of the viral glycoprotein (Env-CT) exhibits a cell-type specific replication phenotype such that replicative spread occurs in some T-cell lines (referred to as permissive cells) but fails to do so in most T-cell lines or in PBMCs (referred to as non-permissive cells). We aim to gain insight on the underlying requirement for the Env-CT for viral spread in non-permissive cells.

Results: We established that in comparison to HIV-Wt, both cell-free and cell-to-cell transmission of mutant HIV-Env-Tr712 from non-permissive cells were severely impaired under naturally low infection conditions. This requirement for Env-CT could be largely overcome by using saturating amounts of virus for infection. We further observed that in permissive cells, which supported both routes of mutant virus transmission, viral gene expression levels, Gag processing and particle release were inherently higher than in non-permissive cells, a factor which may be significantly contributing to their permissivity phenotype. Additionally, and correlating with viral transfer efficiencies in these cell types, HIV-Gag accumulation at the virological synapse (VS) was reduced to background levels in the absence of the Env-CT in conjugates of non-permissive cells but not in permissive cells.

Conclusions: During natural infection conditions, the HIV-Env-CT is critically required for viral transmission in cultures of non-permissive cells by both cell-free and cell-to-cell routes and is instrumental for Gag accumulation to the VS. The requirement of the Env-CT for these related processes is abrogated in permissive cells, which exhibit higher HIV gene expression levels.

Show MeSH
Related in: MedlinePlus