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. 2010 May 12:7:43.
doi: 10.1186/1742-4690-7-43.

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

Vanessa Emerson  1 , Claudia Haller, Tanya Pfeiffer, Oliver T Fackler, Valerie Bosch
Affiliations

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

Vanessa Emerson et al. Retrovirology. .

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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Figures

Figure 1
Figure 1
Replicative spread of HIV-Wt and HIV-Env-Tr712 in different T-cells. A. Schematic depiction of the Env proteins from HIV-Wt and HIV-Env-Tr712 B. Replication kinetics of HIV-Wt (circles) and HIV-Env-Tr712 (triangles) in MT-4 cells (p) and H9 cells (np) below. Cells were infected with equal amounts of virus (equivalent to 100 ng p24 per 106 cells) and washed at 5 h p.i. Newly produced virions released into the culture supernatants at the times indicated were quantified by CA-ELISA. Note that at 3-4 d post infection, MT-4 cells were infected to 100% with both viruses (as established by indirect immunofluorescence) and succumbed to HIV induced cytotoxicity. Infection of H9 cells with HIV-Wt reached 100% at 4-5 d post-infection whereas infection with HIV-Env-Tr712 virions (produced in permissive 293T cells) resulted in initial infection of only a low percentage (< 5%) of cells which subsequently vanished from the culture. The cut-off of the assay lies at 0.01 ng/ml.
Figure 2
Figure 2
Cell-to-cell and cell-free transmission of HIV-Wt and mutant virions. A. FACS analysis of uninfected donor cells and GFP-labelled H9 (np) or dye-labelled MT-4 (p) uninfected target cells. B. Virus transmissions from H9 (np) donor cells highly infected (high) with VSV-G pseudotyped HIV-Wt, HIV-Env-Tr712 or HIV-EnvFus-. Top panels: cell-to-cell transmission. Washed donor cells were adjusted to 50% infection level with uninfected cells and then mixed with a 4-fold excess of H9 target cells. FACS analysis was performed as in A. The percentage target cells infected with HIV-Wt was set at 100% and the levels of transmission of HIV-Env-Tr712 or HIV-EnvFus-, calculated relative to this. Bottom panels: cell-free infection. Equal amounts of released virions from highly infected donor cells were employed to infect susceptible MT-4 cells as described in the Materials and Methods section. At 48 h p.i., the cells were analysed by intracellular p24 FACS. The percentage of cells infected by HIV-Wt (right peak) was set at 100% and the infectivities of HIV-Env-Tr712 and control HIV-Env Fus- calculated relative to this. C. Virus transmissions from H9 (np) donor cells weakly infected (low) with VSV-G pseudotyped HIV-Wt or HIV-Env-Tr712. Washed donor cells were adjusted to 10% infection level with uninfected cells and then mixed with a 9-fold excess of H9 target cells. Further procedures were as in B. D. As in C. except that MT-4 cells (p) were employed both as donor and target cells. E. Mean percentage transmission levels, relative to that of HIV-Wt, of HIV-Env-Tr712 from H9 (np) donor cells infected to high levels (left panel) (12 experiments), H9 (np) donor cells infected to low levels (middle panel) (4 experiments) or MT-4 (p) donor cells infected to low levels (right panel) (4 experiments). The statistical significance of the respective differences is shown (Student's t-test).
Figure 3
Figure 3
Env incorporation into Wt-HIV and HIV-Env-Tr712 virion. Producer H9 (np) cells had been weakly infected (less than 20% infection level). A. Western blot: the top part of the filter has been probed with gp120 antibodies, the middle part with gp41 mAb, Chessie 8 against the Env C-terminal tail (truncated in HIV-Env-Tr712) and the bottom part with p24 mAb. B. Average gp120 incorporation into HIV-Env-Tr712 virions in comparison to HIV-Wt (from 3 independent experiments: individual values 37%, 118%, 83%).
Figure 4
Figure 4
HIV gag gene expression in non-permissive (np) and permissive (p) cells. A. Western blot analysis employing antibodies to HIV-CA and cellular tubulin of equal amounts of lysates of the indicated cell lines infected to equal levels with HIV-Wt or HIV-Env-Tr712. In this experiment, the amounts of virus released into the culture supernatants (determined by HIV-CA ELISA) were 14 ng/ml and 9 ng/ml for HIV-Wt and HIV-Env-Tr712, respectively, produced in H9 (np) cells and 43 ng/ml for both viruses produced in MT-4 (p) cells. B. Western blot analysis as in A of the indicated cell lines infected to equal levels with HIV-Wt. The positions of the detected gp120/gp160, Pr55gag, p24 and cellular tubulin proteins are given on the right.
Figure 5
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.
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References

    1. Jolly C, Sattentau QJ. Retroviral spread by induction of virological synapses. Traffic. 2004;5:643–50. doi: 10.1111/j.1600-0854.2004.00209.x. - DOI - PubMed
    1. Carr JM, Hocking H, Li P, Burrell CJ. Rapid and efficient cell-to-cell transmission of human immunodeficiency virus infection from monocyte-derived macrophages to peripheral blood lymphocytes. Virology. 1999;265:319–29. doi: 10.1006/viro.1999.0047. - DOI - PubMed
    1. Dimitrov DS, Willey RL, Sato H, Chang LJ, Blumenthal R, Martin MA. Quantitation of human immunodeficiency virus type 1 infection kinetics. J Virol. 1993;67:2182–90. - PMC - PubMed
    1. Sato H, Orenstein J, Dimitrov D, Martin M. Cell-to-cell spread of HIV-1 occurs within minutes and may not involve the participation of virus particles. J Virol. 1992;186:712–24. doi: 10.1016/0042-6822(92)90038-Q. - DOI - PubMed
    1. Blanco J, Bosch B, Fernandez-Figueras MT, Barretina J, Clotet B, Este JA. High level of coreceptor-independent HIV transfer induced by contacts between primary CD4 T cells. J Biol Chem. 2004;279:51305–14. doi: 10.1074/jbc.M408547200. - DOI - PubMed

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