ADAR1 is a novel multi targeted anti-HIV-1 cellular protein

Abstract

We examined the antiviral activity of ADAR1 against HIV-1. Our results indicated that ADAR1 in a transfection system inhibited production of viral proteins and infectious HIV-1 in various cell lines including 293T, HeLa, Jurkat T and primary CD4+ T cells, and was active against a number of X4 and R5 HIV-1 of different clades. Further analysis showed that ADAR1 inhibited viral protein synthesis without any effect on viral RNA synthesis. Mutational analysis showed that ADAR1 introduced most of the A-to-G mutations in the rev RNA, in the region of RNA encoding for Rev Response Element (RRE) binding domain and in env RNA. These mutations inhibited the binding of rev to the RRE and inhibited transport of primary transcripts like gag, pol and env from nucleus to cytoplasm resulting in inhibition of viral protein synthesis without any effect on viral RNA synthesis. Furthermore, ADAR1 induced mutations in the env gene inhibited viral infectivity.

Copyright © 2011 Elsevier Inc. All rights reserved.

Figures

Figure 1. Effect of ADAR1 on HIV-1 protein synthesis and infectivity

293T cells were co-transfected with 0.1µg pNL4.3 plasmid and different amounts of ADAR1 plasmid and the control Renilla Luciferase plasmid. HIV-1 production and infectivity were evaluated after 48 h. Viral protein synthesis was monitored by measuring HIV p24 in culture supernatant (Panel A) and intracellular HIV p24 production (Panel B). Results are given as means ± standard deviation. Measurement of extracellular HIV-1 p24 production in transfected Jurkat cells in the absence and presence of increasing amounts of ADAR1 in three independent experiments are shown (Panel C). 293T cells were transfected with 0.1µg pNL4.3, 0.5µg ADAR1 in presence and absence of shRNA 228, shRNA 1724 and 526. Viral protein synthesis was monitored by measuring HIV p24 in culture supernatant 48 h post transfection (Panel D). CD8 depleted PBMC were nucleofected with 2µg of ADAR1. After 48 h cells were infected with 0.5ng/µl and 1ng/µl, of pNL4.3 virus for 2 h. HIV-1 production was evaluated after 72 h using p24 ELISA and calculating the percentage of inhibition of p24 production in the presence of ADAR1. 0.0475ng of p24 value was considered as 100% where 0.5ng/ml virus was added and 0.333ng of p24 value was considered as 100% where 1ng/ml virus was added (Panel E). Production of extracellular HIV-1 p24 in 293T cells co-transfected with HIV-1 89.6, AD8, pIndie C or pNL4.3 plasmids and increasing amount of ADAR1 DNA (Panel F). 3ng HIV-1 p24 equivalent viruses obtained from 293T cells transfected with pNL4.3 alone or together with ADAR1 from Panel B were used to infect TZM-bl cell line (Panel G). Productive infection was monitored after 48 h by measurement of luciferase activity. * indicates there is a significant difference (P

Figure 1. Effect of ADAR1 on HIV-1 protein synthesis and infectivity

293T cells were co-transfected with 0.1µg pNL4.3 plasmid and different amounts of ADAR1 plasmid and the control Renilla Luciferase plasmid. HIV-1 production and infectivity were evaluated after 48 h. Viral protein synthesis was monitored by measuring HIV p24 in culture supernatant (Panel A) and intracellular HIV p24 production (Panel B). Results are given as means ± standard deviation. Measurement of extracellular HIV-1 p24 production in transfected Jurkat cells in the absence and presence of increasing amounts of ADAR1 in three independent experiments are shown (Panel C). 293T cells were transfected with 0.1µg pNL4.3, 0.5µg ADAR1 in presence and absence of shRNA 228, shRNA 1724 and 526. Viral protein synthesis was monitored by measuring HIV p24 in culture supernatant 48 h post transfection (Panel D). CD8 depleted PBMC were nucleofected with 2µg of ADAR1. After 48 h cells were infected with 0.5ng/µl and 1ng/µl, of pNL4.3 virus for 2 h. HIV-1 production was evaluated after 72 h using p24 ELISA and calculating the percentage of inhibition of p24 production in the presence of ADAR1. 0.0475ng of p24 value was considered as 100% where 0.5ng/ml virus was added and 0.333ng of p24 value was considered as 100% where 1ng/ml virus was added (Panel E). Production of extracellular HIV-1 p24 in 293T cells co-transfected with HIV-1 89.6, AD8, pIndie C or pNL4.3 plasmids and increasing amount of ADAR1 DNA (Panel F). 3ng HIV-1 p24 equivalent viruses obtained from 293T cells transfected with pNL4.3 alone or together with ADAR1 from Panel B were used to infect TZM-bl cell line (Panel G). Productive infection was monitored after 48 h by measurement of luciferase activity. * indicates there is a significant difference (P

Figure 2. Expression of endogenous ADAR1 in Jurkat T cell line and its inhibitory capacity on HIV-1 protein synthesis

Jurkat T cells were stimulated with 100units/ml, 500units/ml and 1000 units/ml IFNα for different length of time, 12 h, 24 h, 48 h and 72 h. At each time point cells were harvested and expression of ADAR1 was determined by Western blot using ADAR1 antibody and normalized against β-actin loading control. Relative intensity of ADAR1 p150 in the blot was quantitated (Panel A). Jurkat cells were stimulated with IFN α for 72 h and then infected with 500 ng/ml of pNL4.3 virus. After 48 h viral protein synthesis was monitored by measuring the HIV-1 p24 production in the culture supernatant (Panel B). Jurkat cells were stimulated with IFN α for 72 h. After 72 h cells were transfected with 2µg of shRNA 228 against ADAR1 or control scramble shRNA 526 constructs for 48 h. After 48 h cells were infected with 500 ng/ml of virus. 48 h after infection viral protein synthesis was monitored by measuring the p24 production in the culture supernatant (Panel C).

Figure 2. Expression of endogenous ADAR1 in Jurkat T cell line and its inhibitory capacity on HIV-1 protein synthesis

Jurkat T cells were stimulated with 100units/ml, 500units/ml and 1000 units/ml IFNα for different length of time, 12 h, 24 h, 48 h and 72 h. At each time point cells were harvested and expression of ADAR1 was determined by Western blot using ADAR1 antibody and normalized against β-actin loading control. Relative intensity of ADAR1 p150 in the blot was quantitated (Panel A). Jurkat cells were stimulated with IFN α for 72 h and then infected with 500 ng/ml of pNL4.3 virus. After 48 h viral protein synthesis was monitored by measuring the HIV-1 p24 production in the culture supernatant (Panel B). Jurkat cells were stimulated with IFN α for 72 h. After 72 h cells were transfected with 2µg of shRNA 228 against ADAR1 or control scramble shRNA 526 constructs for 48 h. After 48 h cells were infected with 500 ng/ml of virus. 48 h after infection viral protein synthesis was monitored by measuring the p24 production in the culture supernatant (Panel C).

Figure 3. HIV-1 viral protein synthesis in the presence of ADAR1

293T cells were co-transfected with pNL4.3 plasmid (0.1µg), and ADAR1 (0.7µg) or control empty vector (pcDNA 3.1, 0.7µg). After 48 hours and the supernatant was filtered and centrifuged at 220000 rpm for 1 hour and the pellet was lysed with RIPA buffer. The transfected cells were also lysed with RIPA buffer and were subjected to Western blot analysis. Western blot analysis of viral supernatant was done using HIV-1 infected patient’s antisera (Panel A) and gp120 polyclonal antibody (Panel B). Number below panel B shows the relative amount of gp120 in the supernatant by the band density. Western Blot was also performed using the cell lysate from the same experiment using HIV-1 infected patient’s antisera as an antibody (Panel C) and gp120 polyclonal antibody (Panel D). Number below panel B shows the relative amount of gp120 in the infected cells. Expression of Nef protein in presence and absence of ADAR1 in cell lysate was analyzed by Western Blot using anti-nef antibody (Panel E).

Figure 3. HIV-1 viral protein synthesis in the presence of ADAR1

293T cells were co-transfected with pNL4.3 plasmid (0.1µg), and ADAR1 (0.7µg) or control empty vector (pcDNA 3.1, 0.7µg). After 48 hours and the supernatant was filtered and centrifuged at 220000 rpm for 1 hour and the pellet was lysed with RIPA buffer. The transfected cells were also lysed with RIPA buffer and were subjected to Western blot analysis. Western blot analysis of viral supernatant was done using HIV-1 infected patient’s antisera (Panel A) and gp120 polyclonal antibody (Panel B). Number below panel B shows the relative amount of gp120 in the supernatant by the band density. Western Blot was also performed using the cell lysate from the same experiment using HIV-1 infected patient’s antisera as an antibody (Panel C) and gp120 polyclonal antibody (Panel D). Number below panel B shows the relative amount of gp120 in the infected cells. Expression of Nef protein in presence and absence of ADAR1 in cell lysate was analyzed by Western Blot using anti-nef antibody (Panel E).

Figure 4. Effect of ADAR1 on HIV-1 RNA synthesis

Viral gag, pol and env RNA was quantitated in cells transfected with pNL4.3 in the presence and absence of ADAR1 DNA by Real-time RT-PCR.

Figure 5. ADAR1 induced mutations in the HIV-1 rev RNA

293T cells were transfected with pNL4.3 and the ADAR1 plasmid DNAs. The RNA isolated from transfected cells were subjected to RT PCR amplification. Amplicons were cloned in pcDNA3.1 and 12–25 clones were picked (in the presence and absence of ADAR1) and sequenced. Mutational analysis of the nucleotide sequence of rev region of HIV-1 genome (Panel A). Four sequences are the representative of all clones with identical mutations. Comparison of amino acid sequences of HIV-1 rev region in the presence and absence of ADAR1 (Panel B).

Figure 6. ADAR induced mutations in HIV-1 env gene

Mutational analysis of the env region of HIV-1 genome in the presence and absence of ADAR1. 293T cells were transfected with pc DNA or ADAR1 constructs and RNA was isolated from infected cells. RT-PCR was done and c DNA of env was cloned in pc DNA vector. Each segment of the env gene was cloned individually (Segment 1 (6221 – 6873), Segment 2 (6872–7521), Segment 3 (7522–8187), and Segment 4 (8188–8803). 24 clones were randomly picked from each segment in presence and absence of ADAR1 and sequenced. In case of env 24 sequences were computed into 11 groups.

Figure 7. A-to-G mutation in RRE region in env affects the binding of Rev

293T cells were transfected with 0.6µg pDM128 construct with 8µg wild type rev or mutants rev constructs 1, 2, 3 and 4. Cells were lysed after 48 hours and CAT ELISA assay was performed. Renilla luciferase plasmid was used to normalize the internal transfection efficiency.

Figure 8. A-to-G mutations introduced by ADAR1 affects the export of gag, pol and env RNA transcripts from nucleus to cytoplasm

293T cells were co-transfected with 0.1µg of pNL4.3 and 0.5µg control pc DNA or 0.5µg ADAR1. After 48 hours PARIS kit (Ambion) was used to isolate RNA and protein from cytoplasm and nucleus of transfected cells. Real-time PCR of gag, pol and env RNA was performed using RNA isolated from cytoplasm and nucleus fraction. The ratio of nuclear RNA: cytoplasmic RNA was plotted for both control pcDNA and ADAR1.

Figure 9. A-to-G mutations in the proviral genome at position 6036, 8413 and 8438 affect the synthesis of viral protein

A-to-G mutations were introduced at position 6036, 8413 and 8438 (single, double and triple) using site directed mutagenesis kit (Stratagene). 293T cells were transfected with 0.1µg mutant constructs and Renilla Luciferase plasmid. Viral protein synthesis was monitored by measuring HIV p24 in culture supernatant (Panel A) and intracellular HIV p24 production (Panel B). Viral gag RNA was quantitated in cells transfected with mutant constructs by Real-time RT-PCR (Panel C).

Figure 10. Evaluation of domain of ADAR1 critical for inhibition of HIV-1 protein synthesis and production of infectious virus

Schematic representation of full length human ADAR1 and ADAR1 mutant constructs (Panel A) and their expression (Panel B). 0.1µg of pNL4.3 plasmid was co-transfected with 1µg of ADAR1 and different domain mutant constructs of ADAR1. The Renilla Luciferase expression was used as transfection normalization control (data not shown). Extracellular p24 production in presence and absence of ADAR1 and its mutant constructs (Panel C). Mutational analysis of the rev region of HIV-1 in presence of wild type and ADAR1 mutant constructs (Panel D).