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. 2020 Jun 1;34(7):963-978.
doi: 10.1097/QAD.0000000000002512.

Productive HIV infection in astrocytes can be established via a nonclassical mechanism

Guan-Han Li  1 , Dragan Maric  2 , Eugene O Major  3 , Avindra Nath  1
Affiliations

Productive HIV infection in astrocytes can be established via a nonclassical mechanism

Guan-Han Li et al. AIDS. .

Abstract

Objective: Astrocytes are proposed to be a critical reservoir of HIV in the brain. However, HIV infection of astrocytes is inefficient in vitro except for cell-to-cell transmission from HIV-infected cells. Here, we explore mechanisms by which cell-free HIV bypasses entry and postentry barriers leading to a productive infection.

Methods: HIV infection of astrocytes was investigated by a variety of techniques including transfection of CD4-expressing plasmid, treatment with lysosomotropic agents or using a transwell culture system loaded with HIV-infected lymphocytes. Infection was monitored by HIV-1 p24 in culture supernatants and integrated proviral DNA was quantified by Alu-PCR.

Results: Persistent HIV infection could be established in astrocytes by transfection of proviral DNA, transduction with VSV-G-pseudotyped viruses, transient expression of CD4 followed by HIV infection, or simultaneous treatment with lysosomotropic chloroquine or Tat-HA2 peptide with HIV infection. In absence of these treatments, HIV entered via endocytosis as seen by electronmicroscopy and underwent lysosomal degradation without proviral integration, indicating endocytosis is a dead end for HIV in astrocytes. Nevertheless, productive infection was observed when astrocytes were in close proximity but physically separated from HIV-infected lymphocytes in the transwell cultures. This occurred with X4 or dual tropic R5X4 viruses and was blocked by an antibody or antagonist to CXCR4.

Conclusion: A CD4-independent, CXCR4-dependent mechanism of viral entry is proposed, by which immature HIV particles from infected lymphocytes might directly bind to CXCR4 on astrocytes and trigger virus--cell fusion during or after the process of viral maturation. This mechanism may contribute to the formation of brain HIV reservoirs.

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Conflict of interest statement

CONFLICT OF INTEREST

All authors declare no conflict of interest.

Figures

Fig 1.
Fig 1.. Establishment of persistent HIV-1 infection in primary astrocytes by overcoming the barrier to viral entry.
(A) HIV-1 p24 levels declined rapidly when HFAs were infected with HIV-1 NL4–3 and YK-JRCSF; however, the virus was persistently released into culture medium for at least 2 months when the cells were transfected with pYK-JRCSF or pNLENG1. Persistent infection was also seen when the cells were infected with NL4–3Y’/VSV-G pseudovirus. (B) Significant HIV infection was noted when HFAs were transfected with a plasmid expressing CD4 and then infected with reporter virus NLENG1, however no infection was observed in the cells transfected with an empty vector and then infected with the virus. (C) FACS analysis was performed for HFAs pre-transfected with CD4-expressing plasmid or empty vector and infected with NLENG1. (D) HFAs were treated with ChQ (60 μM) and simultaneously inoculated with NLENG1. The cells were washed next day and maintained in 20 μM ChQ for 5 days, and then fixed and immunostained for GFAP (red fluorescence). Green fluorescence, indicative of HIV infection, was only observed in the ChQ-treated cells. (E) GFP-expressing astrocytes were quantified by FACS following infection with NLENG1 in the presence or absence of ChQ. (F) Using the same experimental paradigm in (D), HFAs were infected with R5 reporter virus, SF162R3. Infection was only observed in the ChQ-treated cells. (G) GFP-expressing astrocytes were quantified by FACS following infection with SF162R3 in the presence or absence of ChQ. (H) Two hours post-infection, HFAs were treated with 60–400 μM ChQ for one day and maintained in 20 μM ChQ for 5 days. The infected cells were quantified by FACS. (I-K) Quantification of HIV-1 p24 in the culture supernatants of HFAs following infection with (I) NLENG1, (J) SF162R3, (K) NL4–3 and IIIB in the presence or absence of ChQ. (L, M) Proviral DNA was measured by qPCR for HFAs simultaneously infected with DNA-free IIIB or 92HT599 in the presence or absence of ChQ. Photo magnification is 200x in (D, F) and 100x in (H).
Fig 1.
Fig 1.. Establishment of persistent HIV-1 infection in primary astrocytes by overcoming the barrier to viral entry.
(A) HIV-1 p24 levels declined rapidly when HFAs were infected with HIV-1 NL4–3 and YK-JRCSF; however, the virus was persistently released into culture medium for at least 2 months when the cells were transfected with pYK-JRCSF or pNLENG1. Persistent infection was also seen when the cells were infected with NL4–3Y’/VSV-G pseudovirus. (B) Significant HIV infection was noted when HFAs were transfected with a plasmid expressing CD4 and then infected with reporter virus NLENG1, however no infection was observed in the cells transfected with an empty vector and then infected with the virus. (C) FACS analysis was performed for HFAs pre-transfected with CD4-expressing plasmid or empty vector and infected with NLENG1. (D) HFAs were treated with ChQ (60 μM) and simultaneously inoculated with NLENG1. The cells were washed next day and maintained in 20 μM ChQ for 5 days, and then fixed and immunostained for GFAP (red fluorescence). Green fluorescence, indicative of HIV infection, was only observed in the ChQ-treated cells. (E) GFP-expressing astrocytes were quantified by FACS following infection with NLENG1 in the presence or absence of ChQ. (F) Using the same experimental paradigm in (D), HFAs were infected with R5 reporter virus, SF162R3. Infection was only observed in the ChQ-treated cells. (G) GFP-expressing astrocytes were quantified by FACS following infection with SF162R3 in the presence or absence of ChQ. (H) Two hours post-infection, HFAs were treated with 60–400 μM ChQ for one day and maintained in 20 μM ChQ for 5 days. The infected cells were quantified by FACS. (I-K) Quantification of HIV-1 p24 in the culture supernatants of HFAs following infection with (I) NLENG1, (J) SF162R3, (K) NL4–3 and IIIB in the presence or absence of ChQ. (L, M) Proviral DNA was measured by qPCR for HFAs simultaneously infected with DNA-free IIIB or 92HT599 in the presence or absence of ChQ. Photo magnification is 200x in (D, F) and 100x in (H).
Fig 1.
Fig 1.. Establishment of persistent HIV-1 infection in primary astrocytes by overcoming the barrier to viral entry.
(A) HIV-1 p24 levels declined rapidly when HFAs were infected with HIV-1 NL4–3 and YK-JRCSF; however, the virus was persistently released into culture medium for at least 2 months when the cells were transfected with pYK-JRCSF or pNLENG1. Persistent infection was also seen when the cells were infected with NL4–3Y’/VSV-G pseudovirus. (B) Significant HIV infection was noted when HFAs were transfected with a plasmid expressing CD4 and then infected with reporter virus NLENG1, however no infection was observed in the cells transfected with an empty vector and then infected with the virus. (C) FACS analysis was performed for HFAs pre-transfected with CD4-expressing plasmid or empty vector and infected with NLENG1. (D) HFAs were treated with ChQ (60 μM) and simultaneously inoculated with NLENG1. The cells were washed next day and maintained in 20 μM ChQ for 5 days, and then fixed and immunostained for GFAP (red fluorescence). Green fluorescence, indicative of HIV infection, was only observed in the ChQ-treated cells. (E) GFP-expressing astrocytes were quantified by FACS following infection with NLENG1 in the presence or absence of ChQ. (F) Using the same experimental paradigm in (D), HFAs were infected with R5 reporter virus, SF162R3. Infection was only observed in the ChQ-treated cells. (G) GFP-expressing astrocytes were quantified by FACS following infection with SF162R3 in the presence or absence of ChQ. (H) Two hours post-infection, HFAs were treated with 60–400 μM ChQ for one day and maintained in 20 μM ChQ for 5 days. The infected cells were quantified by FACS. (I-K) Quantification of HIV-1 p24 in the culture supernatants of HFAs following infection with (I) NLENG1, (J) SF162R3, (K) NL4–3 and IIIB in the presence or absence of ChQ. (L, M) Proviral DNA was measured by qPCR for HFAs simultaneously infected with DNA-free IIIB or 92HT599 in the presence or absence of ChQ. Photo magnification is 200x in (D, F) and 100x in (H).
Fig 1.
Fig 1.. Establishment of persistent HIV-1 infection in primary astrocytes by overcoming the barrier to viral entry.
(A) HIV-1 p24 levels declined rapidly when HFAs were infected with HIV-1 NL4–3 and YK-JRCSF; however, the virus was persistently released into culture medium for at least 2 months when the cells were transfected with pYK-JRCSF or pNLENG1. Persistent infection was also seen when the cells were infected with NL4–3Y’/VSV-G pseudovirus. (B) Significant HIV infection was noted when HFAs were transfected with a plasmid expressing CD4 and then infected with reporter virus NLENG1, however no infection was observed in the cells transfected with an empty vector and then infected with the virus. (C) FACS analysis was performed for HFAs pre-transfected with CD4-expressing plasmid or empty vector and infected with NLENG1. (D) HFAs were treated with ChQ (60 μM) and simultaneously inoculated with NLENG1. The cells were washed next day and maintained in 20 μM ChQ for 5 days, and then fixed and immunostained for GFAP (red fluorescence). Green fluorescence, indicative of HIV infection, was only observed in the ChQ-treated cells. (E) GFP-expressing astrocytes were quantified by FACS following infection with NLENG1 in the presence or absence of ChQ. (F) Using the same experimental paradigm in (D), HFAs were infected with R5 reporter virus, SF162R3. Infection was only observed in the ChQ-treated cells. (G) GFP-expressing astrocytes were quantified by FACS following infection with SF162R3 in the presence or absence of ChQ. (H) Two hours post-infection, HFAs were treated with 60–400 μM ChQ for one day and maintained in 20 μM ChQ for 5 days. The infected cells were quantified by FACS. (I-K) Quantification of HIV-1 p24 in the culture supernatants of HFAs following infection with (I) NLENG1, (J) SF162R3, (K) NL4–3 and IIIB in the presence or absence of ChQ. (L, M) Proviral DNA was measured by qPCR for HFAs simultaneously infected with DNA-free IIIB or 92HT599 in the presence or absence of ChQ. Photo magnification is 200x in (D, F) and 100x in (H).
Fig 1.
Fig 1.. Establishment of persistent HIV-1 infection in primary astrocytes by overcoming the barrier to viral entry.
(A) HIV-1 p24 levels declined rapidly when HFAs were infected with HIV-1 NL4–3 and YK-JRCSF; however, the virus was persistently released into culture medium for at least 2 months when the cells were transfected with pYK-JRCSF or pNLENG1. Persistent infection was also seen when the cells were infected with NL4–3Y’/VSV-G pseudovirus. (B) Significant HIV infection was noted when HFAs were transfected with a plasmid expressing CD4 and then infected with reporter virus NLENG1, however no infection was observed in the cells transfected with an empty vector and then infected with the virus. (C) FACS analysis was performed for HFAs pre-transfected with CD4-expressing plasmid or empty vector and infected with NLENG1. (D) HFAs were treated with ChQ (60 μM) and simultaneously inoculated with NLENG1. The cells were washed next day and maintained in 20 μM ChQ for 5 days, and then fixed and immunostained for GFAP (red fluorescence). Green fluorescence, indicative of HIV infection, was only observed in the ChQ-treated cells. (E) GFP-expressing astrocytes were quantified by FACS following infection with NLENG1 in the presence or absence of ChQ. (F) Using the same experimental paradigm in (D), HFAs were infected with R5 reporter virus, SF162R3. Infection was only observed in the ChQ-treated cells. (G) GFP-expressing astrocytes were quantified by FACS following infection with SF162R3 in the presence or absence of ChQ. (H) Two hours post-infection, HFAs were treated with 60–400 μM ChQ for one day and maintained in 20 μM ChQ for 5 days. The infected cells were quantified by FACS. (I-K) Quantification of HIV-1 p24 in the culture supernatants of HFAs following infection with (I) NLENG1, (J) SF162R3, (K) NL4–3 and IIIB in the presence or absence of ChQ. (L, M) Proviral DNA was measured by qPCR for HFAs simultaneously infected with DNA-free IIIB or 92HT599 in the presence or absence of ChQ. Photo magnification is 200x in (D, F) and 100x in (H).
Fig 1.
Fig 1.. Establishment of persistent HIV-1 infection in primary astrocytes by overcoming the barrier to viral entry.
(A) HIV-1 p24 levels declined rapidly when HFAs were infected with HIV-1 NL4–3 and YK-JRCSF; however, the virus was persistently released into culture medium for at least 2 months when the cells were transfected with pYK-JRCSF or pNLENG1. Persistent infection was also seen when the cells were infected with NL4–3Y’/VSV-G pseudovirus. (B) Significant HIV infection was noted when HFAs were transfected with a plasmid expressing CD4 and then infected with reporter virus NLENG1, however no infection was observed in the cells transfected with an empty vector and then infected with the virus. (C) FACS analysis was performed for HFAs pre-transfected with CD4-expressing plasmid or empty vector and infected with NLENG1. (D) HFAs were treated with ChQ (60 μM) and simultaneously inoculated with NLENG1. The cells were washed next day and maintained in 20 μM ChQ for 5 days, and then fixed and immunostained for GFAP (red fluorescence). Green fluorescence, indicative of HIV infection, was only observed in the ChQ-treated cells. (E) GFP-expressing astrocytes were quantified by FACS following infection with NLENG1 in the presence or absence of ChQ. (F) Using the same experimental paradigm in (D), HFAs were infected with R5 reporter virus, SF162R3. Infection was only observed in the ChQ-treated cells. (G) GFP-expressing astrocytes were quantified by FACS following infection with SF162R3 in the presence or absence of ChQ. (H) Two hours post-infection, HFAs were treated with 60–400 μM ChQ for one day and maintained in 20 μM ChQ for 5 days. The infected cells were quantified by FACS. (I-K) Quantification of HIV-1 p24 in the culture supernatants of HFAs following infection with (I) NLENG1, (J) SF162R3, (K) NL4–3 and IIIB in the presence or absence of ChQ. (L, M) Proviral DNA was measured by qPCR for HFAs simultaneously infected with DNA-free IIIB or 92HT599 in the presence or absence of ChQ. Photo magnification is 200x in (D, F) and 100x in (H).
Fig 2.
Fig 2.. HIV entry by endocytosis and enhanced infection with Tat-HA2 in primary astrocytes.
(A) Increased density on the cell membranes appeared at the sites of HIV attachment to astrocytes (i- iv) and extended to the surrounding area of viral attachment (ii and iii) or formed a crescent border at the site of invagination between the cell membrane and viral particle (iv). (B) HIV particles were partially or completely endocytosed into astrocytes. High-density signal at the site of viral attachment to the cell membrane was seen in the endocytic vesicles (i-iv). A bridge-like connection formed between the viral particle and the cell membrane (iv). (C) HIV particles were observed in the endosome (i) and endolysosomes (ii, iii) of astrocytes (indicated by arrows). (D) Diagrammatic representation of synthetic peptides and their sequences that were used for aiding HIV escape from degradation in the endosomes/lysosomes of astrocytes. (E) Infection of HFAs with NL4–3 was significantly increased by Tat-HA2 in a dose-dependent manner, but not by other peptides. HIV-1 p24 was quantified as described in the previous figures, 3 weeks post-infection. (F-H) Similar results were obtained following infection with (C) NLENG1, (D) YK-JRCSF and (E) SF162R3.
Fig 2.
Fig 2.. HIV entry by endocytosis and enhanced infection with Tat-HA2 in primary astrocytes.
(A) Increased density on the cell membranes appeared at the sites of HIV attachment to astrocytes (i- iv) and extended to the surrounding area of viral attachment (ii and iii) or formed a crescent border at the site of invagination between the cell membrane and viral particle (iv). (B) HIV particles were partially or completely endocytosed into astrocytes. High-density signal at the site of viral attachment to the cell membrane was seen in the endocytic vesicles (i-iv). A bridge-like connection formed between the viral particle and the cell membrane (iv). (C) HIV particles were observed in the endosome (i) and endolysosomes (ii, iii) of astrocytes (indicated by arrows). (D) Diagrammatic representation of synthetic peptides and their sequences that were used for aiding HIV escape from degradation in the endosomes/lysosomes of astrocytes. (E) Infection of HFAs with NL4–3 was significantly increased by Tat-HA2 in a dose-dependent manner, but not by other peptides. HIV-1 p24 was quantified as described in the previous figures, 3 weeks post-infection. (F-H) Similar results were obtained following infection with (C) NLENG1, (D) YK-JRCSF and (E) SF162R3.
Fig 2.
Fig 2.. HIV entry by endocytosis and enhanced infection with Tat-HA2 in primary astrocytes.
(A) Increased density on the cell membranes appeared at the sites of HIV attachment to astrocytes (i- iv) and extended to the surrounding area of viral attachment (ii and iii) or formed a crescent border at the site of invagination between the cell membrane and viral particle (iv). (B) HIV particles were partially or completely endocytosed into astrocytes. High-density signal at the site of viral attachment to the cell membrane was seen in the endocytic vesicles (i-iv). A bridge-like connection formed between the viral particle and the cell membrane (iv). (C) HIV particles were observed in the endosome (i) and endolysosomes (ii, iii) of astrocytes (indicated by arrows). (D) Diagrammatic representation of synthetic peptides and their sequences that were used for aiding HIV escape from degradation in the endosomes/lysosomes of astrocytes. (E) Infection of HFAs with NL4–3 was significantly increased by Tat-HA2 in a dose-dependent manner, but not by other peptides. HIV-1 p24 was quantified as described in the previous figures, 3 weeks post-infection. (F-H) Similar results were obtained following infection with (C) NLENG1, (D) YK-JRCSF and (E) SF162R3.
Fig 2.
Fig 2.. HIV entry by endocytosis and enhanced infection with Tat-HA2 in primary astrocytes.
(A) Increased density on the cell membranes appeared at the sites of HIV attachment to astrocytes (i- iv) and extended to the surrounding area of viral attachment (ii and iii) or formed a crescent border at the site of invagination between the cell membrane and viral particle (iv). (B) HIV particles were partially or completely endocytosed into astrocytes. High-density signal at the site of viral attachment to the cell membrane was seen in the endocytic vesicles (i-iv). A bridge-like connection formed between the viral particle and the cell membrane (iv). (C) HIV particles were observed in the endosome (i) and endolysosomes (ii, iii) of astrocytes (indicated by arrows). (D) Diagrammatic representation of synthetic peptides and their sequences that were used for aiding HIV escape from degradation in the endosomes/lysosomes of astrocytes. (E) Infection of HFAs with NL4–3 was significantly increased by Tat-HA2 in a dose-dependent manner, but not by other peptides. HIV-1 p24 was quantified as described in the previous figures, 3 weeks post-infection. (F-H) Similar results were obtained following infection with (C) NLENG1, (D) YK-JRCSF and (E) SF162R3.
Fig 3.
Fig 3.. Low level of CD4 expression and regulation of CD4, CXCR4 mRNA by pro-inflammatory cytokines in primary astrocytes.
(A-D) mRNA levels for (A) CD4, (B) CXCR4, (C) CCR5 and (D) DC-SIGN were measured in astrocytes from different sources and compared to monocytic (THP-1) and lymphocytic (Jurkat E6–1) cell lines. PDA, progenitor-derived astrocytes. HFA “E”, “M”, “H22359” were from different fetuses; HFA “1”, “2”, “3” were prepared from different batches of H22359. (E) CD4 was detected by Western blot analysis in T cell lines JKT and Jurkat E6 cells (left), and by immunoprecipitation (IP) with anti-CD4 antibody or IgG isotype control in lysates of Jurkat E6–1 and HEK 293 cells prepared from 2 × 106 cells each (middle) and in lysates of PDAs and HFAs prepared from 1× 106 cells (right). (F) HFAs were exposed to a panel of cytokines either individually or in various combinations at concentrations of 10 ng/ml each for 3 days. mRNA level of CD4 was significantly upregulated by IFN-γ individually or when combined with TNF-α or/and IL-1β. (G) The cells were treated as described in (F); mRNA level of CXCR4 was downregulated by IFN-γ but significantly upregulated by the combination of IFN-γ, TNF-α and IL-1β. Data represents mean ± SEM from at least three experiments. (H) No significant enhancement of HIV infection was observed in the astrocytes that were pre-treated with a combination of IFN-γ, TNF-α, IL-1β (10 ng/ml for each) for 3 days and then infected with NLENG1 or 89.6 and monitored for 21 days. (I) No significant increase of HIV infection was observed in the astrocytes that were pre-treated with a combination of IFN-γ, TNF-α, IL-1β (10 ng/ml for each) for 3 days and then infected with multiple HIV-1 strains and monitored for 21 days, compared to HFA controls. (J) CD4 protein was not increased in the astrocytes following treatment with 10 ng/ml of IFN-γ, TNF-α and IL-1β for 3 days when cell lysates were analyzed by Western blot or IP. Western blot was performed using 40 μg of cell lysate in each lane and IP was performed using 1000 μg of cell lysate for each sample. (K) CD4 and CXCR4 were analyzed by FACS in live HFAs 2 days post-treatment with or without IFN-γ, TNF-α, IL-1β (10 ng/ml for each). ANOVA with unequal variance and Dunnett’s method was used for statistical analysis. * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001, ***** p<0.0001.
Fig 3.
Fig 3.. Low level of CD4 expression and regulation of CD4, CXCR4 mRNA by pro-inflammatory cytokines in primary astrocytes.
(A-D) mRNA levels for (A) CD4, (B) CXCR4, (C) CCR5 and (D) DC-SIGN were measured in astrocytes from different sources and compared to monocytic (THP-1) and lymphocytic (Jurkat E6–1) cell lines. PDA, progenitor-derived astrocytes. HFA “E”, “M”, “H22359” were from different fetuses; HFA “1”, “2”, “3” were prepared from different batches of H22359. (E) CD4 was detected by Western blot analysis in T cell lines JKT and Jurkat E6 cells (left), and by immunoprecipitation (IP) with anti-CD4 antibody or IgG isotype control in lysates of Jurkat E6–1 and HEK 293 cells prepared from 2 × 106 cells each (middle) and in lysates of PDAs and HFAs prepared from 1× 106 cells (right). (F) HFAs were exposed to a panel of cytokines either individually or in various combinations at concentrations of 10 ng/ml each for 3 days. mRNA level of CD4 was significantly upregulated by IFN-γ individually or when combined with TNF-α or/and IL-1β. (G) The cells were treated as described in (F); mRNA level of CXCR4 was downregulated by IFN-γ but significantly upregulated by the combination of IFN-γ, TNF-α and IL-1β. Data represents mean ± SEM from at least three experiments. (H) No significant enhancement of HIV infection was observed in the astrocytes that were pre-treated with a combination of IFN-γ, TNF-α, IL-1β (10 ng/ml for each) for 3 days and then infected with NLENG1 or 89.6 and monitored for 21 days. (I) No significant increase of HIV infection was observed in the astrocytes that were pre-treated with a combination of IFN-γ, TNF-α, IL-1β (10 ng/ml for each) for 3 days and then infected with multiple HIV-1 strains and monitored for 21 days, compared to HFA controls. (J) CD4 protein was not increased in the astrocytes following treatment with 10 ng/ml of IFN-γ, TNF-α and IL-1β for 3 days when cell lysates were analyzed by Western blot or IP. Western blot was performed using 40 μg of cell lysate in each lane and IP was performed using 1000 μg of cell lysate for each sample. (K) CD4 and CXCR4 were analyzed by FACS in live HFAs 2 days post-treatment with or without IFN-γ, TNF-α, IL-1β (10 ng/ml for each). ANOVA with unequal variance and Dunnett’s method was used for statistical analysis. * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001, ***** p<0.0001.
Fig 3.
Fig 3.. Low level of CD4 expression and regulation of CD4, CXCR4 mRNA by pro-inflammatory cytokines in primary astrocytes.
(A-D) mRNA levels for (A) CD4, (B) CXCR4, (C) CCR5 and (D) DC-SIGN were measured in astrocytes from different sources and compared to monocytic (THP-1) and lymphocytic (Jurkat E6–1) cell lines. PDA, progenitor-derived astrocytes. HFA “E”, “M”, “H22359” were from different fetuses; HFA “1”, “2”, “3” were prepared from different batches of H22359. (E) CD4 was detected by Western blot analysis in T cell lines JKT and Jurkat E6 cells (left), and by immunoprecipitation (IP) with anti-CD4 antibody or IgG isotype control in lysates of Jurkat E6–1 and HEK 293 cells prepared from 2 × 106 cells each (middle) and in lysates of PDAs and HFAs prepared from 1× 106 cells (right). (F) HFAs were exposed to a panel of cytokines either individually or in various combinations at concentrations of 10 ng/ml each for 3 days. mRNA level of CD4 was significantly upregulated by IFN-γ individually or when combined with TNF-α or/and IL-1β. (G) The cells were treated as described in (F); mRNA level of CXCR4 was downregulated by IFN-γ but significantly upregulated by the combination of IFN-γ, TNF-α and IL-1β. Data represents mean ± SEM from at least three experiments. (H) No significant enhancement of HIV infection was observed in the astrocytes that were pre-treated with a combination of IFN-γ, TNF-α, IL-1β (10 ng/ml for each) for 3 days and then infected with NLENG1 or 89.6 and monitored for 21 days. (I) No significant increase of HIV infection was observed in the astrocytes that were pre-treated with a combination of IFN-γ, TNF-α, IL-1β (10 ng/ml for each) for 3 days and then infected with multiple HIV-1 strains and monitored for 21 days, compared to HFA controls. (J) CD4 protein was not increased in the astrocytes following treatment with 10 ng/ml of IFN-γ, TNF-α and IL-1β for 3 days when cell lysates were analyzed by Western blot or IP. Western blot was performed using 40 μg of cell lysate in each lane and IP was performed using 1000 μg of cell lysate for each sample. (K) CD4 and CXCR4 were analyzed by FACS in live HFAs 2 days post-treatment with or without IFN-γ, TNF-α, IL-1β (10 ng/ml for each). ANOVA with unequal variance and Dunnett’s method was used for statistical analysis. * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001, ***** p<0.0001.
Fig 3.
Fig 3.. Low level of CD4 expression and regulation of CD4, CXCR4 mRNA by pro-inflammatory cytokines in primary astrocytes.
(A-D) mRNA levels for (A) CD4, (B) CXCR4, (C) CCR5 and (D) DC-SIGN were measured in astrocytes from different sources and compared to monocytic (THP-1) and lymphocytic (Jurkat E6–1) cell lines. PDA, progenitor-derived astrocytes. HFA “E”, “M”, “H22359” were from different fetuses; HFA “1”, “2”, “3” were prepared from different batches of H22359. (E) CD4 was detected by Western blot analysis in T cell lines JKT and Jurkat E6 cells (left), and by immunoprecipitation (IP) with anti-CD4 antibody or IgG isotype control in lysates of Jurkat E6–1 and HEK 293 cells prepared from 2 × 106 cells each (middle) and in lysates of PDAs and HFAs prepared from 1× 106 cells (right). (F) HFAs were exposed to a panel of cytokines either individually or in various combinations at concentrations of 10 ng/ml each for 3 days. mRNA level of CD4 was significantly upregulated by IFN-γ individually or when combined with TNF-α or/and IL-1β. (G) The cells were treated as described in (F); mRNA level of CXCR4 was downregulated by IFN-γ but significantly upregulated by the combination of IFN-γ, TNF-α and IL-1β. Data represents mean ± SEM from at least three experiments. (H) No significant enhancement of HIV infection was observed in the astrocytes that were pre-treated with a combination of IFN-γ, TNF-α, IL-1β (10 ng/ml for each) for 3 days and then infected with NLENG1 or 89.6 and monitored for 21 days. (I) No significant increase of HIV infection was observed in the astrocytes that were pre-treated with a combination of IFN-γ, TNF-α, IL-1β (10 ng/ml for each) for 3 days and then infected with multiple HIV-1 strains and monitored for 21 days, compared to HFA controls. (J) CD4 protein was not increased in the astrocytes following treatment with 10 ng/ml of IFN-γ, TNF-α and IL-1β for 3 days when cell lysates were analyzed by Western blot or IP. Western blot was performed using 40 μg of cell lysate in each lane and IP was performed using 1000 μg of cell lysate for each sample. (K) CD4 and CXCR4 were analyzed by FACS in live HFAs 2 days post-treatment with or without IFN-γ, TNF-α, IL-1β (10 ng/ml for each). ANOVA with unequal variance and Dunnett’s method was used for statistical analysis. * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001, ***** p<0.0001.
Fig 3.
Fig 3.. Low level of CD4 expression and regulation of CD4, CXCR4 mRNA by pro-inflammatory cytokines in primary astrocytes.
(A-D) mRNA levels for (A) CD4, (B) CXCR4, (C) CCR5 and (D) DC-SIGN were measured in astrocytes from different sources and compared to monocytic (THP-1) and lymphocytic (Jurkat E6–1) cell lines. PDA, progenitor-derived astrocytes. HFA “E”, “M”, “H22359” were from different fetuses; HFA “1”, “2”, “3” were prepared from different batches of H22359. (E) CD4 was detected by Western blot analysis in T cell lines JKT and Jurkat E6 cells (left), and by immunoprecipitation (IP) with anti-CD4 antibody or IgG isotype control in lysates of Jurkat E6–1 and HEK 293 cells prepared from 2 × 106 cells each (middle) and in lysates of PDAs and HFAs prepared from 1× 106 cells (right). (F) HFAs were exposed to a panel of cytokines either individually or in various combinations at concentrations of 10 ng/ml each for 3 days. mRNA level of CD4 was significantly upregulated by IFN-γ individually or when combined with TNF-α or/and IL-1β. (G) The cells were treated as described in (F); mRNA level of CXCR4 was downregulated by IFN-γ but significantly upregulated by the combination of IFN-γ, TNF-α and IL-1β. Data represents mean ± SEM from at least three experiments. (H) No significant enhancement of HIV infection was observed in the astrocytes that were pre-treated with a combination of IFN-γ, TNF-α, IL-1β (10 ng/ml for each) for 3 days and then infected with NLENG1 or 89.6 and monitored for 21 days. (I) No significant increase of HIV infection was observed in the astrocytes that were pre-treated with a combination of IFN-γ, TNF-α, IL-1β (10 ng/ml for each) for 3 days and then infected with multiple HIV-1 strains and monitored for 21 days, compared to HFA controls. (J) CD4 protein was not increased in the astrocytes following treatment with 10 ng/ml of IFN-γ, TNF-α and IL-1β for 3 days when cell lysates were analyzed by Western blot or IP. Western blot was performed using 40 μg of cell lysate in each lane and IP was performed using 1000 μg of cell lysate for each sample. (K) CD4 and CXCR4 were analyzed by FACS in live HFAs 2 days post-treatment with or without IFN-γ, TNF-α, IL-1β (10 ng/ml for each). ANOVA with unequal variance and Dunnett’s method was used for statistical analysis. * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001, ***** p<0.0001.
Fig 4.
Fig 4.. Establishment of productive HIV infection in primary astrocytes or U373 MG cell lines via the transwell culture system.
(A) A transwell culture device was used to determine if newly produced HIV particles could infect astrocytes. Astrocytes were pre-seeded in the wells of culture plate and HIV-infected lymphocytes were seeded in the transwell inserts, the membrane of which has a pore size of 0.4 μm. (B) NLENG1-infected HFAs were observed by 3–5 days post-transwell culture. The infected astrocytes were immunostained for glial fibrillary acid protein (GFAP). (C) 20 μg/ml of antibodies to CD4, CXCR4 or DC-SIGN were pre-incubated with HFAs and maintained during the transwell culture. Each insert was loaded with 4 × 105 NLENG1-infected JKT cells. 3 days post-transwell culture, the inserts were removed and the astrocytes were washed with PBS and replenished with fresh culture medium. HIV-1 p24 was measured in the culture media at different time points. (D) Antibodies to CD4, CXCR4 or DC-SIGN; fusion inhibitor T20 (500 nM) and CXCR4 antagonist AMD3100 (50–100 μM) were used to determine their effects on HIV infection of astrocytes in the transwell cultures. The inserts were removed 3–5 days post-transwell culture and HIV-1 p24 was measured in the culture media after 3–4 weeks. Data represent mean ± SEM from three experiments and was analyzed by one-way ANOVA. * p<0.05, ** p<0.01, *** p<0.005. (E) Similar experiments were performed to test whether other HIV-1 strains were also able to infect primary astrocytes via transwell cultures. The inserts were removed 5 days post-transwell culture. The results represent mean ± SEM from three experiments. (F) Proviral DNA was measured by Alu-qPCR in HFAs collected from 6-well plates of the transwell cultures where IIIB- or 92HT599-infected JKTs were placed in the top-chambers for 4–5 days. The HFAs were at least washed for three times with DPBS before collection. (G) Four cell lines derived from U373 MG were generated: U373 MG_1–6 (CD4−, CXCR4−), U373 MG_1–8 (CD4+, CXCR4−), U373 MG_2–4 (CD4−, CXCR4+) and U373 MG_2–10 (CD4+, CXCR4+). Expression of CD4 and CXCR4 on cell membrane was measured by FACS. (H) EGFP was strongly expressed in U373 MG_2–10 after 4 days of infection by both cell-free virus and the transwell culture, indicating significant infection with NLENG1. (I) HIV infection was observed in U373 MG_2–4 with only the use of transwell cultures, but no significant infection was seen in the cells with cell-free virus 4 days post-infection. (J) HIV-1 p24 was measured over time in the supernatants of U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 infected with cell-free virus. The cells were initially infected with the viral supernatant collected from NLENG1-infected JKT cells (with 4 days-infection) and washed 3 times with DPBS 3 days post-infection. (K) Levels of HIV-1 p24 were measured over 3 weeks in U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 infected by the transwell culture. The transwell inserts were removed 3 days post-infection and the cells were washed 3 times with DPBS and replenished with fresh culture medium. (L) The ratio of HIV-1 p24 levels of the infection by the transwell cultures to those of the infection with cell-free virus in U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 was ploted over time. Photo magnification is 200x in (B, H) and 100x in (I).
Fig 4.
Fig 4.. Establishment of productive HIV infection in primary astrocytes or U373 MG cell lines via the transwell culture system.
(A) A transwell culture device was used to determine if newly produced HIV particles could infect astrocytes. Astrocytes were pre-seeded in the wells of culture plate and HIV-infected lymphocytes were seeded in the transwell inserts, the membrane of which has a pore size of 0.4 μm. (B) NLENG1-infected HFAs were observed by 3–5 days post-transwell culture. The infected astrocytes were immunostained for glial fibrillary acid protein (GFAP). (C) 20 μg/ml of antibodies to CD4, CXCR4 or DC-SIGN were pre-incubated with HFAs and maintained during the transwell culture. Each insert was loaded with 4 × 105 NLENG1-infected JKT cells. 3 days post-transwell culture, the inserts were removed and the astrocytes were washed with PBS and replenished with fresh culture medium. HIV-1 p24 was measured in the culture media at different time points. (D) Antibodies to CD4, CXCR4 or DC-SIGN; fusion inhibitor T20 (500 nM) and CXCR4 antagonist AMD3100 (50–100 μM) were used to determine their effects on HIV infection of astrocytes in the transwell cultures. The inserts were removed 3–5 days post-transwell culture and HIV-1 p24 was measured in the culture media after 3–4 weeks. Data represent mean ± SEM from three experiments and was analyzed by one-way ANOVA. * p<0.05, ** p<0.01, *** p<0.005. (E) Similar experiments were performed to test whether other HIV-1 strains were also able to infect primary astrocytes via transwell cultures. The inserts were removed 5 days post-transwell culture. The results represent mean ± SEM from three experiments. (F) Proviral DNA was measured by Alu-qPCR in HFAs collected from 6-well plates of the transwell cultures where IIIB- or 92HT599-infected JKTs were placed in the top-chambers for 4–5 days. The HFAs were at least washed for three times with DPBS before collection. (G) Four cell lines derived from U373 MG were generated: U373 MG_1–6 (CD4−, CXCR4−), U373 MG_1–8 (CD4+, CXCR4−), U373 MG_2–4 (CD4−, CXCR4+) and U373 MG_2–10 (CD4+, CXCR4+). Expression of CD4 and CXCR4 on cell membrane was measured by FACS. (H) EGFP was strongly expressed in U373 MG_2–10 after 4 days of infection by both cell-free virus and the transwell culture, indicating significant infection with NLENG1. (I) HIV infection was observed in U373 MG_2–4 with only the use of transwell cultures, but no significant infection was seen in the cells with cell-free virus 4 days post-infection. (J) HIV-1 p24 was measured over time in the supernatants of U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 infected with cell-free virus. The cells were initially infected with the viral supernatant collected from NLENG1-infected JKT cells (with 4 days-infection) and washed 3 times with DPBS 3 days post-infection. (K) Levels of HIV-1 p24 were measured over 3 weeks in U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 infected by the transwell culture. The transwell inserts were removed 3 days post-infection and the cells were washed 3 times with DPBS and replenished with fresh culture medium. (L) The ratio of HIV-1 p24 levels of the infection by the transwell cultures to those of the infection with cell-free virus in U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 was ploted over time. Photo magnification is 200x in (B, H) and 100x in (I).
Fig 4.
Fig 4.. Establishment of productive HIV infection in primary astrocytes or U373 MG cell lines via the transwell culture system.
(A) A transwell culture device was used to determine if newly produced HIV particles could infect astrocytes. Astrocytes were pre-seeded in the wells of culture plate and HIV-infected lymphocytes were seeded in the transwell inserts, the membrane of which has a pore size of 0.4 μm. (B) NLENG1-infected HFAs were observed by 3–5 days post-transwell culture. The infected astrocytes were immunostained for glial fibrillary acid protein (GFAP). (C) 20 μg/ml of antibodies to CD4, CXCR4 or DC-SIGN were pre-incubated with HFAs and maintained during the transwell culture. Each insert was loaded with 4 × 105 NLENG1-infected JKT cells. 3 days post-transwell culture, the inserts were removed and the astrocytes were washed with PBS and replenished with fresh culture medium. HIV-1 p24 was measured in the culture media at different time points. (D) Antibodies to CD4, CXCR4 or DC-SIGN; fusion inhibitor T20 (500 nM) and CXCR4 antagonist AMD3100 (50–100 μM) were used to determine their effects on HIV infection of astrocytes in the transwell cultures. The inserts were removed 3–5 days post-transwell culture and HIV-1 p24 was measured in the culture media after 3–4 weeks. Data represent mean ± SEM from three experiments and was analyzed by one-way ANOVA. * p<0.05, ** p<0.01, *** p<0.005. (E) Similar experiments were performed to test whether other HIV-1 strains were also able to infect primary astrocytes via transwell cultures. The inserts were removed 5 days post-transwell culture. The results represent mean ± SEM from three experiments. (F) Proviral DNA was measured by Alu-qPCR in HFAs collected from 6-well plates of the transwell cultures where IIIB- or 92HT599-infected JKTs were placed in the top-chambers for 4–5 days. The HFAs were at least washed for three times with DPBS before collection. (G) Four cell lines derived from U373 MG were generated: U373 MG_1–6 (CD4−, CXCR4−), U373 MG_1–8 (CD4+, CXCR4−), U373 MG_2–4 (CD4−, CXCR4+) and U373 MG_2–10 (CD4+, CXCR4+). Expression of CD4 and CXCR4 on cell membrane was measured by FACS. (H) EGFP was strongly expressed in U373 MG_2–10 after 4 days of infection by both cell-free virus and the transwell culture, indicating significant infection with NLENG1. (I) HIV infection was observed in U373 MG_2–4 with only the use of transwell cultures, but no significant infection was seen in the cells with cell-free virus 4 days post-infection. (J) HIV-1 p24 was measured over time in the supernatants of U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 infected with cell-free virus. The cells were initially infected with the viral supernatant collected from NLENG1-infected JKT cells (with 4 days-infection) and washed 3 times with DPBS 3 days post-infection. (K) Levels of HIV-1 p24 were measured over 3 weeks in U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 infected by the transwell culture. The transwell inserts were removed 3 days post-infection and the cells were washed 3 times with DPBS and replenished with fresh culture medium. (L) The ratio of HIV-1 p24 levels of the infection by the transwell cultures to those of the infection with cell-free virus in U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 was ploted over time. Photo magnification is 200x in (B, H) and 100x in (I).
Fig 4.
Fig 4.. Establishment of productive HIV infection in primary astrocytes or U373 MG cell lines via the transwell culture system.
(A) A transwell culture device was used to determine if newly produced HIV particles could infect astrocytes. Astrocytes were pre-seeded in the wells of culture plate and HIV-infected lymphocytes were seeded in the transwell inserts, the membrane of which has a pore size of 0.4 μm. (B) NLENG1-infected HFAs were observed by 3–5 days post-transwell culture. The infected astrocytes were immunostained for glial fibrillary acid protein (GFAP). (C) 20 μg/ml of antibodies to CD4, CXCR4 or DC-SIGN were pre-incubated with HFAs and maintained during the transwell culture. Each insert was loaded with 4 × 105 NLENG1-infected JKT cells. 3 days post-transwell culture, the inserts were removed and the astrocytes were washed with PBS and replenished with fresh culture medium. HIV-1 p24 was measured in the culture media at different time points. (D) Antibodies to CD4, CXCR4 or DC-SIGN; fusion inhibitor T20 (500 nM) and CXCR4 antagonist AMD3100 (50–100 μM) were used to determine their effects on HIV infection of astrocytes in the transwell cultures. The inserts were removed 3–5 days post-transwell culture and HIV-1 p24 was measured in the culture media after 3–4 weeks. Data represent mean ± SEM from three experiments and was analyzed by one-way ANOVA. * p<0.05, ** p<0.01, *** p<0.005. (E) Similar experiments were performed to test whether other HIV-1 strains were also able to infect primary astrocytes via transwell cultures. The inserts were removed 5 days post-transwell culture. The results represent mean ± SEM from three experiments. (F) Proviral DNA was measured by Alu-qPCR in HFAs collected from 6-well plates of the transwell cultures where IIIB- or 92HT599-infected JKTs were placed in the top-chambers for 4–5 days. The HFAs were at least washed for three times with DPBS before collection. (G) Four cell lines derived from U373 MG were generated: U373 MG_1–6 (CD4−, CXCR4−), U373 MG_1–8 (CD4+, CXCR4−), U373 MG_2–4 (CD4−, CXCR4+) and U373 MG_2–10 (CD4+, CXCR4+). Expression of CD4 and CXCR4 on cell membrane was measured by FACS. (H) EGFP was strongly expressed in U373 MG_2–10 after 4 days of infection by both cell-free virus and the transwell culture, indicating significant infection with NLENG1. (I) HIV infection was observed in U373 MG_2–4 with only the use of transwell cultures, but no significant infection was seen in the cells with cell-free virus 4 days post-infection. (J) HIV-1 p24 was measured over time in the supernatants of U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 infected with cell-free virus. The cells were initially infected with the viral supernatant collected from NLENG1-infected JKT cells (with 4 days-infection) and washed 3 times with DPBS 3 days post-infection. (K) Levels of HIV-1 p24 were measured over 3 weeks in U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 infected by the transwell culture. The transwell inserts were removed 3 days post-infection and the cells were washed 3 times with DPBS and replenished with fresh culture medium. (L) The ratio of HIV-1 p24 levels of the infection by the transwell cultures to those of the infection with cell-free virus in U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 was ploted over time. Photo magnification is 200x in (B, H) and 100x in (I).
Fig 4.
Fig 4.. Establishment of productive HIV infection in primary astrocytes or U373 MG cell lines via the transwell culture system.
(A) A transwell culture device was used to determine if newly produced HIV particles could infect astrocytes. Astrocytes were pre-seeded in the wells of culture plate and HIV-infected lymphocytes were seeded in the transwell inserts, the membrane of which has a pore size of 0.4 μm. (B) NLENG1-infected HFAs were observed by 3–5 days post-transwell culture. The infected astrocytes were immunostained for glial fibrillary acid protein (GFAP). (C) 20 μg/ml of antibodies to CD4, CXCR4 or DC-SIGN were pre-incubated with HFAs and maintained during the transwell culture. Each insert was loaded with 4 × 105 NLENG1-infected JKT cells. 3 days post-transwell culture, the inserts were removed and the astrocytes were washed with PBS and replenished with fresh culture medium. HIV-1 p24 was measured in the culture media at different time points. (D) Antibodies to CD4, CXCR4 or DC-SIGN; fusion inhibitor T20 (500 nM) and CXCR4 antagonist AMD3100 (50–100 μM) were used to determine their effects on HIV infection of astrocytes in the transwell cultures. The inserts were removed 3–5 days post-transwell culture and HIV-1 p24 was measured in the culture media after 3–4 weeks. Data represent mean ± SEM from three experiments and was analyzed by one-way ANOVA. * p<0.05, ** p<0.01, *** p<0.005. (E) Similar experiments were performed to test whether other HIV-1 strains were also able to infect primary astrocytes via transwell cultures. The inserts were removed 5 days post-transwell culture. The results represent mean ± SEM from three experiments. (F) Proviral DNA was measured by Alu-qPCR in HFAs collected from 6-well plates of the transwell cultures where IIIB- or 92HT599-infected JKTs were placed in the top-chambers for 4–5 days. The HFAs were at least washed for three times with DPBS before collection. (G) Four cell lines derived from U373 MG were generated: U373 MG_1–6 (CD4−, CXCR4−), U373 MG_1–8 (CD4+, CXCR4−), U373 MG_2–4 (CD4−, CXCR4+) and U373 MG_2–10 (CD4+, CXCR4+). Expression of CD4 and CXCR4 on cell membrane was measured by FACS. (H) EGFP was strongly expressed in U373 MG_2–10 after 4 days of infection by both cell-free virus and the transwell culture, indicating significant infection with NLENG1. (I) HIV infection was observed in U373 MG_2–4 with only the use of transwell cultures, but no significant infection was seen in the cells with cell-free virus 4 days post-infection. (J) HIV-1 p24 was measured over time in the supernatants of U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 infected with cell-free virus. The cells were initially infected with the viral supernatant collected from NLENG1-infected JKT cells (with 4 days-infection) and washed 3 times with DPBS 3 days post-infection. (K) Levels of HIV-1 p24 were measured over 3 weeks in U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 infected by the transwell culture. The transwell inserts were removed 3 days post-infection and the cells were washed 3 times with DPBS and replenished with fresh culture medium. (L) The ratio of HIV-1 p24 levels of the infection by the transwell cultures to those of the infection with cell-free virus in U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 was ploted over time. Photo magnification is 200x in (B, H) and 100x in (I).
Fig 4.
Fig 4.. Establishment of productive HIV infection in primary astrocytes or U373 MG cell lines via the transwell culture system.
(A) A transwell culture device was used to determine if newly produced HIV particles could infect astrocytes. Astrocytes were pre-seeded in the wells of culture plate and HIV-infected lymphocytes were seeded in the transwell inserts, the membrane of which has a pore size of 0.4 μm. (B) NLENG1-infected HFAs were observed by 3–5 days post-transwell culture. The infected astrocytes were immunostained for glial fibrillary acid protein (GFAP). (C) 20 μg/ml of antibodies to CD4, CXCR4 or DC-SIGN were pre-incubated with HFAs and maintained during the transwell culture. Each insert was loaded with 4 × 105 NLENG1-infected JKT cells. 3 days post-transwell culture, the inserts were removed and the astrocytes were washed with PBS and replenished with fresh culture medium. HIV-1 p24 was measured in the culture media at different time points. (D) Antibodies to CD4, CXCR4 or DC-SIGN; fusion inhibitor T20 (500 nM) and CXCR4 antagonist AMD3100 (50–100 μM) were used to determine their effects on HIV infection of astrocytes in the transwell cultures. The inserts were removed 3–5 days post-transwell culture and HIV-1 p24 was measured in the culture media after 3–4 weeks. Data represent mean ± SEM from three experiments and was analyzed by one-way ANOVA. * p<0.05, ** p<0.01, *** p<0.005. (E) Similar experiments were performed to test whether other HIV-1 strains were also able to infect primary astrocytes via transwell cultures. The inserts were removed 5 days post-transwell culture. The results represent mean ± SEM from three experiments. (F) Proviral DNA was measured by Alu-qPCR in HFAs collected from 6-well plates of the transwell cultures where IIIB- or 92HT599-infected JKTs were placed in the top-chambers for 4–5 days. The HFAs were at least washed for three times with DPBS before collection. (G) Four cell lines derived from U373 MG were generated: U373 MG_1–6 (CD4−, CXCR4−), U373 MG_1–8 (CD4+, CXCR4−), U373 MG_2–4 (CD4−, CXCR4+) and U373 MG_2–10 (CD4+, CXCR4+). Expression of CD4 and CXCR4 on cell membrane was measured by FACS. (H) EGFP was strongly expressed in U373 MG_2–10 after 4 days of infection by both cell-free virus and the transwell culture, indicating significant infection with NLENG1. (I) HIV infection was observed in U373 MG_2–4 with only the use of transwell cultures, but no significant infection was seen in the cells with cell-free virus 4 days post-infection. (J) HIV-1 p24 was measured over time in the supernatants of U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 infected with cell-free virus. The cells were initially infected with the viral supernatant collected from NLENG1-infected JKT cells (with 4 days-infection) and washed 3 times with DPBS 3 days post-infection. (K) Levels of HIV-1 p24 were measured over 3 weeks in U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 infected by the transwell culture. The transwell inserts were removed 3 days post-infection and the cells were washed 3 times with DPBS and replenished with fresh culture medium. (L) The ratio of HIV-1 p24 levels of the infection by the transwell cultures to those of the infection with cell-free virus in U373 MG_1–6, U373 MG_1–8 and U373 MG_2–4 was ploted over time. Photo magnification is 200x in (B, H) and 100x in (I).
Fig. 5.
Fig. 5.. Model of HIV infection in human astrocytes.
(A) Cell-free, mature HIV attaches to astrocyte via an unspecified receptor resulting in endocytosis. The virus is retained in the endosome or endolysosome and finally gets degraded there. (B) CXCR4-binding sites on the envelope of immature HIV may be in an “open” state that allows the virus to directly bind to CXCR4 on the cell membrane of astrocytes, and hidden following a conformational change that is triggered during HIV maturation. The virus that was pre-bound in an immature state finally triggers the fusion process between HIV envelope and the astrocyte membrane during or after the process of viral maturation, leading to HIV infection of astrocytes in a CD4-independent but CXCR4-dependent manner. This cannot occur with cell-free, mature HIV because astrocytes lack sufficient expression of CD4 on the membrane and CXCR4-binding sites are hidden in the envelope of viral particles.
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